CN109957569B - Base editing system and method based on CPF1 protein - Google Patents

Abstract

The present invention relates to the field of genetic engineering. Specifically, the invention relates to a base editing method based on CPF1 protein. More particularly, the present invention relates to a method for efficient base editing of a target sequence in the genome of an organism (e.g., a plant) by a guide RNA-directed Cpf 1-deaminase fusion protein, and genetically modified organisms (e.g., plants) and progeny thereof produced by said method.

Description

CPF1 protein-based base editing system and method

Technical Field

The present invention relates to the field of genetic engineering. In particular, the invention relates to a base editing system and method based on CPF1 protein. More particularly, the present invention relates to a system and method for efficient base editing of a target sequence in the genome of an organism (e.g., a plant) by a guide RNA-directed Cpf 1-deaminase fusion protein, and genetically modified organisms (e.g., plants) and progeny thereof produced by the method.

Background

A prerequisite for efficient crop improvement is the availability of new genetic mutations, which can be readily introduced into modern cultivars. Genetic studies, especially based on genome-wide association, have shown that single nucleotide changes are a major cause of differences in crop traits. Single base variation leads to amino acid substitutions, resulting in the evolution of superior alleles and superior traits. Targeted induction of local mutations in the genome (TILLING) before genome editing has occurred may serve as a means for generating mutations that are urgently needed in crop improvement. However, TILLING screening is time and labor consuming, and the point mutations identified are often limited in number and type. Genome editing techniques, particularly those based on the CRISPR/Cas9 system, can achieve the introduction of substitutions of specific bases in genomic sites through Homologous Recombination (HR) -mediated DNA repair pathways. However, at present, the successful use of this method is greatly limited, mainly due to the low frequency with which HR-mediated double-strand break repair occurs in plants. In addition, it is currently a major challenge to efficiently provide sufficient quantities of DNA repair templates. These problems make it challenging to achieve site-directed mutagenesis efficiently and simply in plants by way of HR.

In recent years, by fusing Cas9 and deaminase using the binding property of Cas9 and DNA and the property of DNA deaminase, it is possible to realize the conversion of the single base cytosine (C) to thymine (T) and the conversion of adenine (a) to guanine (G) of a target gene with high precision. Currently, systems for C to T transitions include mainly fusions of SpnCas9-BE3, spnCas9-AID and Cas9 variants such as VQR-BE3, EQR-BE3 and VRER-BE3, in addition to SaCas9-BE3 and the variant SaKKH-BE3, which combinations reduce the PAM limit of cytosine (C) to thymine (T) transitions and the extent of the editing window is more variable. In addition, adenine deaminase capable of acting on ssDNA is developed by David Liu laboratories of Harvard university recently in an artificial evolution mode, and a Cas9-ABE system capable of realizing A-to-G conversion of DNA by fusion with Cas9 is developed, so that the action direction of single base editing is further expanded. Although these studies have led to a wide range of uses for single base editing of DNA, there are still a number of problems with current single base editing techniques: first, since PAM for Cas9 and Cas9 variants is generally limited to G/C rich regions, PAM types for single base editing systems still need to be broadened; secondly, as the specificity of Cas9 and editing itself is poor, the single base editing system still needs to be improved in the aspect of specificity; thirdly, since the nCas9-BE3 and variant BE3 thereof, and nCas9-ABE usually generate single-stranded nicks on the non-targeting strand of the target site, it is easy to generate single-base mutation and DNA fidelity and insertion during mismatch repair, so that the high fidelity of single base is also to BE improved. Thus, there remains a great need in the art for new systems and methods for base editing plant genomes.

Description of the drawings

FIG. 1 optimization of CPF1-mediated plant genome cleavage activity.

Cpf 1-mediated mutation of plant genomes C to T.

Cpf 1-mediated mutation of plant genomes a to G.

FIG. 4 simultaneous base editing at multiple sites using the RNA cleavage activity of CPF1.

Disclosure of Invention

1. Definition of

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures, as used herein, are all terms and routine procedures used extensively in the relevant arts. For example, standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following references: sambrook, j., fritsch, e.f. and manitis, t., molecular Cloning: (iii) an Arabidopsis Manual; cold Spring Harbor Laboratory Press: cold Spring Harbor,1989 (hereinafter referred to as "Sambrook"). Meanwhile, in order to better understand the present invention, the following provides definitions and explanations of related terms.

"Cpf1 nuclease," "Cpf1 protein," and "Cpf1" are used interchangeably herein to refer to RNA-guided nucleases comprising a Cpf1 protein or fragment thereof. Cpf1 is a component of the CRISPR-Cpf1 genome editing system and is capable of targeting and cleaving a DNA target sequence under the direction of a guide RNA (crRNA) to form a DNA Double Strand Break (DSB). The Cpf1 protein comprises a DNA cleavage domain and an independent RNA cleavage domain. The RNA cleavage domain of the Cpf1 protein is capable of processing pre-crRNA to form mature crRNA.

"guide RNA" and "gRNA" are used interchangeably herein. The guide RNA of the Cpf 1-mediated genome editing system typically consists only of mature crRNA molecules, wherein the crRNA comprises a sequence that is sufficiently identical to the target sequence to hybridize to the complement of the target sequence and direct specific binding of the complex (Cpf 1+ crRNA) to the target sequence.

"deaminase" refers to an enzyme that catalyzes a deamination reaction. In some embodiments of the invention, the deaminase refers to a cytosine deaminase that catalyzes the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments of the invention, the deaminase refers to an adenine deaminase that is capable of catalyzing the formation of inosine (I) from adenosine or deoxyadenosine (a).

"genome" when used in a plant cell encompasses not only chromosomal DNA present in the nucleus, but organelle DNA present in subcellular components of the cell (e.g., mitochondria, plastids).

As used herein, "organism" includes any organism suitable for genome editing, preferably a eukaryote. Examples of organisms include, but are not limited to, mammals such as mice, rats, monkeys, dogs, pigs, sheep, cows, cats; poultry such as chicken, duck, goose; plants include both monocots and dicots, such as rice, maize, wheat, sorghum, barley, soybean, peanut, arabidopsis, and the like.

By "genetically modified organism" or "genetically modified cell" is meant an organism or cell that comprises within its genome an exogenous polynucleotide or modified gene or expression control sequence. For example, an exogenous polynucleotide can be stably integrated into the genome of an organism or cell and be inherited by successive generations. The exogenous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct. Modified genes or expression control sequences are those which comprise single or multiple deoxynucleotide substitutions, deletions and additions in the genome of an organism or cell. For example, the genetically modified organism obtained by the present invention may comprise one or more C to T or a to G substitutions relative to the wild type (corresponding organism not genetically modified).

"exogenous" with respect to a sequence means a sequence from a foreign species, or if from the same species, a sequence whose composition and/or locus has been significantly altered from its native form by deliberate human intervention.

"polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and are single-or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides are referred to by their single letter designation as follows: "A" is adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

"polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

As used herein, "expression construct" refers to a vector, such as a recombinant vector, suitable for expression of a nucleotide sequence of interest in an organism. "expression" refers to the production of a functional product. For example, expression of a nucleotide sequence can refer to transcription of the nucleotide sequence (e.g., transcription to produce mRNA or functional RNA) and/or translation of the RNA into a precursor or mature protein.

The "expression construct" of the invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, may be an RNA (e.g., mRNA) capable of translation.

An "expression construct" of the invention may comprise regulatory sequences and nucleotide sequences of interest of different origin, or regulatory sequences and nucleotide sequences of interest of the same origin but arranged in a manner different from that normally found in nature.

"regulatory sequence" and "regulatory element" are used interchangeably to refer to a nucleotide sequence that is located upstream (5 'non-coding sequence), intermediate, or downstream (3' non-coding sequence) of a coding sequence and that affects the transcription, RNA processing or stability, or translation of the associated coding sequence.

Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

"promoter" refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the invention, the promoter is a promoter capable of controlling transcription of a gene in a cell of an organism, whether or not it is derived from the organism. The promoter may be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.

"constitutive promoter" refers to a promoter that will generally cause a gene to be expressed in most cell types under most circumstances. "tissue-specific promoter" and "tissue-preferred promoter" are used interchangeably and refer to a promoter that is expressed primarily, but not necessarily exclusively, in a tissue or organ, but may also be expressed in a particular cell or cell type. "developmentally regulated promoter" refers to a promoter whose activity is determined by a developmental event. An "inducible promoter" selectively expresses an operably linked DNA sequence in response to an endogenous or exogenous stimulus (environmental, hormonal, chemical signal, etc.).

As used herein, the term "operably linked" refers to a regulatory element (such as, but not limited to, a promoter sequence, a transcription termination sequence, and the like) linked to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

"introducing" a nucleic acid molecule (e.g., a plasmid, linear nucleic acid fragment, RNA, etc.) or a protein into an organism refers to transforming cells of the organism with the nucleic acid or protein such that the nucleic acid or protein is capable of functioning in the cells. "transformation" as used herein includes both stable transformation and transient transformation.

"Stable transformation" refers to the introduction of an exogenous nucleotide sequence into a genome, resulting in the stable inheritance of the exogenous gene. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generation thereof.

"transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell that performs a function without stable inheritance of a foreign gene. In transient transformation, the foreign nucleic acid sequence is not integrated into the genome.

As used herein, the term "plant" includes whole plants and any progeny, cell, tissue, or part of a plant. The term "plant part" includes any part of a plant, including, for example, but not limited to: seeds (including mature seeds, immature embryos without seed coats, and immature seeds); plant cutting; a plant cell; a plant cell culture; plant organs (e.g., pollen, embryos, flowers, fruits, buds, leaves, roots, stems, and related explants). The plant tissue or plant organ may be a seed, callus, or any other population of plant cells organized into structural or functional units. The plant cell or tissue culture is capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was derived, and is capable of regenerating a plant having substantially the same genotype as the plant. In contrast, some plant cells are not capable of regenerating to produce plants. The regenerable cells in the plant cell or tissue culture can be embryos, protoplasts, meristematic cells, callus tissue, pollen, leaves, anthers, roots, root tips, silks, flowers, kernels, ears, cobs, shells, or stems.

Plant parts include harvestable parts and parts useful for propagating progeny plants. Plant parts useful for propagation include, for example, but are not limited to: seeds; fruits; cutting; seedling; a tuber; and a rootstock. Harvestable parts of a plant may be any useful part of a plant, including, for example, but not limited to: flower; pollen; seedling; a tuber; leaves; a stem; fruits; seeds; and a root.

Plant cells are the structural and physiological units of plants. As used herein, plant cells include protoplasts and protoplasts having a partial cell wall. Plant cells may be in the form of isolated individual cells or cell aggregates (e.g., loose callus and cultured cells), and may be part of a higher order tissue unit (e.g., plant tissue, plant organs, and plants). Thus, a plant cell may be a protoplast, a gamete producing cell, or a cell or collection of cells capable of regenerating into a whole plant. Thus, in embodiments herein, a seed comprising a plurality of plant cells and capable of regenerating into a whole plant is considered a "plant part".

As used herein, the term "protoplast" refers to a plant cell with a completely or partially removed cell wall and an uncovered lipid bilayer membrane. Typically, protoplasts are isolated plant cells without a cell wall, which have the potential to regenerate into cell cultures or whole plants.

Plant "progeny" includes any subsequent generation of the plant.

"trait" refers to a physiological, morphological, biochemical or physical characteristic of a plant or a particular plant material or cell. In some embodiments, these characteristics may be visible to the naked eye, such as the seed, size of the plant, etc.; indexes that can be measured by biochemical techniques, such as the content of protein, starch or oil in seeds or leaves; observable metabolic or physiological processes, such as determining resistance to water stress, specific salt, sugar or nitrogen concentrations; a detectable level of gene expression; or agronomic traits such as resistance to osmotic stress or yield can be observed. In some embodiments, the trait also includes the ploidy (ploidy) of the plant, such as the haploidy (ploidy) important for plant breeding. In some embodiments, the trait further comprises resistance of the plant to a herbicide.

An "agronomic trait" is a measurable indicator parameter including, but not limited to: leaf green, grain yield, growth rate, total biomass or accumulation rate, fresh weight at maturity, dry weight at maturity, fruit yield, seed yield, plant total nitrogen content, fruit nitrogen content, seed nitrogen content, plant vegetative tissue nitrogen content, plant total free amino acid content, fruit free amino acid content, seed free amino acid content, plant vegetative tissue free amino acid content, plant total protein content, fruit protein content, seed protein content, plant vegetative tissue protein content, drought resistance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear length, disease resistance, cold resistance, salt resistance, tiller number, and the like.

2.Cpf1 protein-based base editing system

The present invention provides a system for base editing of a target sequence in a genome of an organism, comprising at least one of the following i) to v):

i) Base-editing fusion proteins, and guide RNAs;

ii) an expression construct comprising a nucleotide sequence encoding a base-editing fusion protein, and a guide RNA;

iii) A base-editing fusion protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;

iv) an expression construct comprising a nucleotide sequence encoding a base-editing fusion protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;

v) an expression construct comprising a nucleotide sequence encoding a base-editing fusion protein and a nucleotide sequence encoding a guide RNA;

wherein the base-editing fusion protein comprises Cpf1 lacking DNA cleavage activity and a deaminase, the guide RNA being capable of targeting the base-editing fusion protein to a target sequence in a genome resulting in a substitution of one or more of C to T or A to G in the target sequence.

Cpf1 contains a DNA cleavage domain (RuvC) which can be mutated to delete the DNA cleavage activity of Cpf1, resulting in "Cpf1 with deleted DNA cleavage activity". Cpf1 with the loss of DNA cleavage activity still retained gRNA-directed DNA binding ability. Thus, in principle, cpf1 lacking DNA cleavage activity may, when fused to another protein, target said other protein to almost any DNA sequence simply by co-expression with a suitable guide RNA.

Cpf1 deleted for DNA cleavage activity according to the invention may be derived from different species of Cpf1, for example the Cpf1 proteins designated FnCpf1 (for example the wild-type amino acid sequence is shown in SEQ ID NO: 19), asCpf1 (for example the wild-type amino acid sequence is shown in SEQ ID NO: 18) and LbCpf1 (for example the wild-type amino acid sequence is shown in SEQ ID NO: 20) derived from Francisella novicida U112, acylaminococcus sp.BV3L6 and Lachnospiraceae bacterium ND2006, respectively.

In some embodiments, the Cpf1 with DNA cleavage activity deletion is FnCpf1 with DNA cleavage activity deletion. In some embodiments, the FnCpf1 lacking DNA cleavage activity comprises a D917A mutation relative to wild-type FnCpf1.

In some embodiments, the Cpf1 deficient in DNA cleaving activity is AsCpf1 deficient in DNA cleaving activity. In some embodiments, the AsCpf1 lacking DNA cleavage activity comprises a D908A mutation relative to wild-type AsCpf1.

In some preferred embodiments, the DNA cleavage activity deleted Cpf1 is DNA cleavage activity deleted LbCpf1. In some embodiments, the LbCpf1 lacking DNA cleavage activity comprises a D832A mutation relative to wild-type LbCpf1.

In some embodiments, the Cpf1 with the deletion of DNA cleavage activity retains its RNA cleavage activity, thereby enabling processing of pre-crRNA to form mature crRNA. Thus, in some embodiments, an expression construct comprising a nucleotide sequence encoding a guide RNA in a system of the invention may comprise a sequence encoding a plurality of different guide RNA (crRNA) precursors in tandem, which upon transcription may be processed by the Cpf1 lacking the DNA cleavage activity to form a plurality of different guide RNAs (crrnas), thereby targeting a plurality of different target sequences simultaneously.

In some embodiments of the invention, the deaminase in the fusion protein is a cytidine deaminase, such as an apolipoprotein B mRNA editing complex (APOBEC) family deaminase.

Cytidine deaminase can catalyze the deamination of cytidine (C) on DNA to form uracil (U). The present inventors have surprisingly found that fusing Cpf1 with a deletion in DNA cleavage activity to a cytidine deaminase allows the fusion protein to target a target sequence in the genome under the guidance of a guide RNA, without cleaving the DNA double strand due to the inactivating deletion in DNA cleavage activity of Cpf1, whereas the cytidine deaminase in the fusion protein allows to deaminate the cytidine of the single stranded DNA generated in the formation of the Cpf 1-guide RNA-DNA complex into U, which in turn allows for C to T substitutions by base mismatch repair.

The cytidine deaminase of the present invention is particularly a cytidine deaminase that can accept single-stranded DNA as a substrate. Examples of cytidine deaminases useful in the present invention include, but are not limited to: APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G or CDA1. In some embodiments of the invention, the cytidine deaminase comprises the amino acid sequence set forth in SEQ ID NO. 1.

In the case where the deaminase in the fusion protein is a cytidine deaminase, the base editing system of the present invention can mutate one or more cs in the genomic target sequence to T, also referred to as the Cpf1-PBE system.

In cells, uracil DNA glycosylase catalyzes the removal of U from DNA and initiates Base Excision Repair (BER), resulting in the repair of U: G to C: G. Thus, without being bound by any theory, the inclusion of a uracil DNA glycosylase inhibitor in a base editing fusion protein of the invention or a system of the invention will enable an increase in the efficiency of base editing.

Thus, in some embodiments of the present invention directed to a Cpf1-PBE system, the base-editing fusion protein further comprises a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the uracil DNA glycosylase inhibitor comprises the amino acid sequence set forth in SEQ ID NO 2.

In some embodiments of the invention, the deaminase is an adenine deaminase.

Naturally occurring adenine deaminases are commonly used with RNA as a substrate to convert adenosine on single-stranded RNA to inosine (I) by deamination. Recently, DNA-dependent adenine deaminases capable of converting deoxyguanosine on a single-stranded DNA into inosine (I) using a single-stranded DNA as a substrate have been obtained based on the tRNA adenine deaminase TadA of Escherichia coli by directed evolution. See Nicloe m. Gaudelli et al, doi:10.1038/nature24644, 2017.

The present inventors have surprisingly found that fusion of Cpf1 with a deletion in DNA cleavage activity, under the direction of guide RNA, to a DNA-dependent adenine deaminase, which can target a target sequence in a plant genome, DNA double strand not being cleaved due to the deletion in DNA cleavage activity of Cpf1, can deaminate adenosine of single stranded DNA generated in Cpf 1-guide RNA-DNA complex formation into inosine (I), which can be substituted a to G by base mismatch repair, since the DNA polymerase would treat inosine (I) as guanine (G). Thus, in the case where the deaminase in the fusion protein is a DNA-dependent adenine deaminase, the base editing system of the invention can mutate one or more a's in the genomic target sequence to G, also referred to as the Cpf1-ABE system.

In some embodiments of the invention, the DNA-dependent adenine deaminase is a variant of escherichia coli tRNA adenine deaminase TadA (ecTadA), in particular a variant that can accept single-stranded DNA as substrate, comprising one or more sets of mutations relative to wild-type ecTadA selected from the group consisting of:

1) A106V and D108N;

2) D147Y and E155V;

3) L84F, H123Y and I156F;

4)A142N；

5) H36L, R51L, S146C and K157N;

6)P48S/T/A；

7)A142N；

8)W23L/R；

9)R152H/P。

in a specific embodiment of the invention, said DNA-dependent adenine deaminase (ABE version 7.9) comprises the following mutations relative to wild-type ecTadA: W23R, H36L, R51L, S146C, K157N, A106V, D108N, P48A, L84F, H123Y, I156F, A142N, D147Y, E155V, and R152P.

In a specific embodiment of the invention, said DNA-dependent adenine deaminase (ABE version 7.10) comprises the following mutations relative to wild-type ecTadA: W23R, H36L, R51L, S146C, K157N, A106V, D108N, P48A, L84F, H123Y, I156F, D147Y, E155V, and R152P.

The wild-type ecTadA amino acid sequence is shown below: <xnotran> MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 3). </xnotran> In some embodiments, wherein the initial methionine may be absent.

Preferred ecTadA-derived DNA-dependent adenine deaminase (ABE version 7.10) amino acid sequences are shown below: <xnotran> MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 4). </xnotran> In some embodiments, wherein the initial methionine may be absent.

In some embodiments of the invention, the deaminase is fused to the N-terminus of Cpf1 where the DNA cleaving activity is absent. In some embodiments, the deaminase is fused to the C-terminus of Cpf1 in which the DNA cleaving activity is absent.

In some preferred embodiments, the DNA-dependent adenine deaminase is fused to the N-terminus of the corresponding wild-type adenine deaminase. It is expected that the formation of heterodimers of DNA-dependent adenine deaminase with wild-type adenine deaminase can significantly improve the editing activity of fusion proteins A to G.

In some embodiments of the invention, the deaminase and the Cpf1 lacking DNA cleavage activity are fused via a linker. The linker may be a non-functional amino acid sequence of 1 to 50 (e.g., 1,2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 20 to 25, 25 to 50) or more amino acids in length, without secondary or more structure. For example, the linker may be a flexible linker, such as GGGGS, GS, GAP, (GGGGS) x3, GGS, and (GGS) x7, and the like. In some specific embodiments, the linker is an XTEN linker. In some embodiments, the linker is 32 amino acids in length. In some specific embodiments, the amino acid sequence of the linker is: SGGSSGSGSETPGTSESATPESSGGSSGGS.

In some embodiments of the invention, the base-editing fusion protein of the invention further comprises a Nuclear Localization Sequence (NLS). In general, the one or more NLS in the base-editing fusion protein should be of sufficient strength to drive the base-editing fusion protein in the nucleus of the plant cell to accumulate in an amount that can perform its base-editing function. In general, the intensity of the nuclear localization activity is determined by the number, location, one or more specific NLS used, or a combination of these factors in the base-editing fusion protein.

In some embodiments of the invention, the NLS of the base-editing fusion protein of the invention may be located at the N-terminus and/or the C-terminus. In some embodiments, the base-editing fusion protein comprises about 1,2, 3, 4, 5, 6, 7, 8,9, 10, or more NLSs. In some embodiments, the base-editing fusion protein comprises about 1,2, 3, 4, 5, 6, 7, 8,9, 10, or more NLSs at or near the N-terminus. In some embodiments, the base-editing fusion protein comprises about 1,2, 3, 4, 5, 6, 7, 8,9, 10 or more NLS at or near the C-terminus. In some embodiments, the base-editing fusion protein comprises a combination of these, such as comprising one or more NLS at the N-terminus and one or more NLS at the C-terminus. When there is more than one NLS, each can be chosen to be independent of the other NLS. In some preferred embodiments of the invention, the base-editing fusion protein comprises 2NLS, e.g. the 2NLS are at the N-terminus and C-terminus, respectively.

In general, NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the surface of the protein, but other types of NLS are also known. Non-limiting examples of NLS include: KKRKV (nucleotide sequence 5-.

In some embodiments of the invention, the N-terminus of the base-editing fusion protein comprises an NLS of the amino acid sequence shown by PKKKRKV. In some embodiments of the invention, the C-terminus of the base-editing fusion protein comprises an NLS of the amino acid sequence set forth in SGGSPKKKRKV or krpaatkkagqakkkkk.

In addition, the base-editing fusion protein of the present invention may further include other localization sequences, such as a cytoplasmic localization sequence, a chloroplast localization sequence, a mitochondrial localization sequence, and the like, depending on the position of the DNA to be edited.

In some embodiments of the invention involving a Cpf1-PBE system, the base-editing fusion protein further comprises uracil DNA glycosylase inhibitor (UGI), and further followed by two NLS at the N-terminus or C-terminus of the UGI. In some preferred embodiments, the base-editing fusion proteins of the invention comprise an amino acid sequence selected from the group consisting of SEQ ID NOS 24-29.

In order to obtain efficient expression, in some embodiments of the invention, the nucleotide sequence encoding the base-editing fusion protein is codon optimized for the biological species to be base-edited.

Codon optimization refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one Codon of the native sequence with a Codon that is more frequently or most frequently used in the gene of the host cell (e.g., about or more than about 1,2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons while maintaining the native amino acid sequence. Different species exhibit a specific preference for certain codons of a particular amino acid. Codon preferences (differences in Codon Usage between organisms) are often related to the translation efficiency of messenger RNA (mRNA) which is believed to be dependent on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules.

In some embodiments, the base-editing fusion protein of the invention is encoded by a nucleotide sequence selected from SEQ ID NOS 8-9, 11-12, or 14-15.

In some embodiments of the invention, the nucleotide sequence encoding the base-editing fusion protein and/or the nucleotide sequence encoding the guide RNA is operably linked to an expression control element, such as a promoter.

Examples of promoters that may be used in the present invention include, but are not limited to, polymerase (pol) I, pol II, or pol III promoters. Examples of pol I promoters include chicken RNA pol I promoters. Examples of pol II promoters include, but are not limited to, the cytomegalovirus immediate early (CMV) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, and the Simian Virus 40 (SV 40) immediate early promoter. Examples of pol III promoters include the U6 and H1 promoters. Inducible promoters such as the metallothionein promoter can be used. Other examples of promoters include the T7 phage promoter, the T3 phage promoter, the β -galactosidase promoter, and the Sp6 phage promoter. When used in plants, the promoter may be a cauliflower mosaic virus 35S promoter, a maize Ubi-1 promoter, a wheat U6 promoter, a rice U3 promoter, a maize U3 promoter, a rice actin promoter.

Preferably, the guide RNA (crRNA) is expressed using the Ubi-1 promoter and cleaved to maturity with a ribozyme such as the HDV ribozyme.

In one embodiment, the addition of an intron after the Ubi-1 promoter can enhance the expression of the protein or RNA of interest.

In some embodiments, the expression construct for expressing the base fusion protein of the invention comprises an expression cassette as set forth in SEQ ID NO.10 or 13. Or the expression construct comprises an expression control sequence shown as SEQ ID NO. 30.

3. Method for producing genetically modified organisms

In another aspect, the invention provides a method of producing a genetically modified organism (such as a plant) comprising introducing into a cell of the organism the system for base editing of a target sequence in the genome of an organism of the invention, whereby the guide RNA targets the base editing fusion protein to the target sequence in the genome of the plant, resulting in the substitution of one or more C's by T's or one or more a's by G's in the target sequence.

The design of a target sequence or crRNA coding sequence that can be recognized and targeted by the Cpf1 protein and guide RNA (i.e., crRNA) complex can be found, for example, in Zhang et al, cell 163,1-13, october 22,2015. In general, the 5' end of the target sequence targeted by the genome editing system of the present invention will desirably comprise a pro-spacer adjacent motif (PAM) 5' -TTTN or 5' -YTN, where N is independently selected from A, G, C and T and Y is selected from C and T.

For example, in some embodiments of the invention, the target sequence has the following structure: 5' -TTTN-N _X -3 'or 5' -YTN-N _X -3', wherein N is independently selected from a, G, C and T, Y is selected from C and T; x is an integer which is more than or equal to 15 and less than or equal to 35; nx represents X consecutive nucleotides.

In the present invention, the target sequence to be modified may be located anywhere in the genome, for example within a functional gene such as a protein-encoding gene, or may be located, for example, in a gene expression regulatory region such as a promoter region or enhancer region, thereby effecting modification of the function of the gene or modification of gene expression.

A to G or C to T base edits in the cell target sequence can be detected by T7EI, PCR/RE or sequencing methods.

In the method of the present invention, the system for base editing can be introduced into a cell by various methods well known to those skilled in the art. Methods that can be used to introduce the genome editing system of the present invention into a cell include, but are not limited to: calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, viral infection (such as baculovirus, vaccinia, adenovirus, adeno-associated virus, lentivirus and other viruses), particle gun methods, PEG-mediated transformation of protoplasts, agrobacterium tumefaciens-mediated transformation.

The cells whose genome can be edited by the method of the present invention may be derived from, for example, mammals such as mouse, rat, monkey, dog, pig, sheep, cow, cat; poultry such as chicken, duck, goose; plants, including monocots and dicots, such as rice, maize, wheat, sorghum, barley, soybean, peanut, arabidopsis, and the like.

The method of the invention is particularly suitable for producing genetically modified plants, such as crop plants. In the method of producing a genetically modified plant of the present invention, the base editing system may be introduced into a plant in various ways well known to those skilled in the art. Methods that can be used to introduce the base editing system of the present invention into a plant include, but are not limited to: particle gun method, PEG mediated protoplast transformation, agrobacterium tumefaciens mediated transformation, plant virus mediated transformation, pollen tube channel method and ovary injection method.

In the method of producing a genetically modified plant of the present invention, modification of a target sequence can be achieved by introducing or producing the base-editing fusion protein and guide RNA into a plant cell, and the modification can be stably inherited without stably transforming a plant with the base-editing system. This avoids the potential off-target effects of the stably existing base editing system and also avoids the integration of exogenous nucleotide sequences in the plant genome, thus having higher biosafety.

In some preferred embodiments, the introduction is performed in the absence of selective pressure, thereby avoiding integration of the exogenous nucleotide sequence in the plant genome.

In some embodiments, the introducing comprises transforming an isolated plant cell or tissue with the base editing system of the invention and then regenerating the transformed plant cell or tissue into a whole plant. Preferably, the regeneration is carried out in the absence of selective pressure, i.e., without using any selective agent for the selection gene carried on the expression vector during the tissue culture process. The regeneration efficiency of the plant can be improved without using a selection agent, and a modified plant without an exogenous nucleotide sequence can be obtained.

In other embodiments, the base editing system of the invention can be transformed into a specific site on an intact plant, such as a leaf, stem tip, pollen tube, young ear, or hypocotyl. This is particularly suitable for the transformation of plants which are difficult to regenerate by tissue culture.

In some embodiments of the invention, the in vitro expressed protein and/or in vitro transcribed RNA molecule is directly transformed into the plant. The protein and/or RNA molecules are capable of effecting base editing in plant cells and subsequent degradation by the cells, avoiding integration of foreign nucleotide sequences in the plant genome.

Plants that can be base edited by the methods of the invention include monocots and dicots. For example, the plant may be a crop plant, such as wheat, rice, maize, soybean, sunflower, sorghum, canola, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, or potato.

In some embodiments of the invention, wherein the target sequence is associated with a plant trait, such as an agronomic trait, whereby the base editing results in the plant having an altered trait relative to a wild type plant.

In the present invention, the target sequence to be modified may be located anywhere in the genome, for example, within a functional gene such as a protein-encoding gene, or may be located, for example, in a gene expression regulatory region such as a promoter region or an enhancer region, thereby effecting a modification of the function of the gene or a modification of gene expression. Accordingly, in some embodiments of the invention, the C to T or a to G substitution results in an amino acid substitution in the target protein or truncation of the target protein (resulting in a stop codon). In other embodiments of the invention, the substitution of C to T or a to G results in a change in expression of the target gene.

In some embodiments of the invention, the method further comprises obtaining progeny of the genetically modified plant.

In another aspect, the present invention also provides a genetically modified plant or progeny or parts thereof, wherein the plant is obtained by the method of the invention as described above.

In another aspect, the present invention also provides a method of breeding a plant, comprising crossing a first genetically modified plant obtained by the above-described method of the present invention with a second plant not containing said genetic modification, thereby introducing said genetic modification into the second plant.

Examples

Construction of Ubi-CPF1-PBE/ABE expression vector

The ABE, XTEN, dCPF1 sequences were codon optimized for plants and ordered from GenScript (Nanjing). The full-length dCPF1-ABE fragment was amplified using the primer pair HindIII-F (with HindIII restriction site) and EcoRI (with EcoRI restriction site). The PCR product was digested with HindIII and EcoRI and then inserted into the two enzyme-digested pJIT163-GFP vector (the vector sequence is shown in SEQ ID NO: 16) to produce a fusion expression vector dCPF1-ABE.

The PBE, XTEN, dCPF1 sequences were codon optimized for plants and ordered from GenScript (Nanjing). The full-length dCPF1-PBE fragment was amplified using the primer pair HindIII-F (with HindIII restriction site) and EcoRI (with EcoRI restriction site). The PCR product was digested with HindIII and EcoRI and then inserted into the two enzyme-digested pJIT163-GFP vector (the vector sequence is shown in SEQ ID NO: 16) to produce a fusion expression vector dCPF1-PBE.

Construction of sgRNA expression vector

According to the previous description (Wang, Y.et al, simultaneous edition of three homo alloys in hexagonal branched coal compositions with a variable resistance to a pore chemistry of Nat. Biotechnol.32,947-951, shan, Q.et al, targeted genome modification of crop plants using a CRISPR-Cas system of Nat. Biotechnol.31,686-688,2013; and Liang, Z.et al.Targeted mutagenesis in Zea Mays using TALENs and the CRISPR/Cas system.J Genet genomics.41,63-68, 2014) construct sgRNA expression vectors based on pTaU6-sgRNA (Addgene ID 53062) or pOsU3-sgRNA (Addgene ID 53063) or pZmU3-sgRNA (Addgene ID 5306) or OsU3/TaU6-tRNA-sgRNA (Zhang et al.2017.genome biology.DOI:10.1186/s 13059-017-1325-9). In addition, crRNA is produced by promoting hammerhead enzyme and crRNA through type II promoter (Tang et al. Nature plant, doi: 10.1038/nplants.2017.18)

pUbi-mGFPP-crRNA、pUbi-DEP1-sgRNA、pUbi-DEP1-crRNA、pUbi-DME-crRNA.

BFP and GFP expression vectors

pUbi-mGFP, the vector sequence is shown in SEQ ID NO:17.

protoplast assay

In this study, a wheat Bobwhite variety and a rice Nipponbare variety were used. Protoplast transformation was performed as follows. The average conversion efficiency was 55-70%. Each plasmid was transformed with 10. Mu.g by PEG-mediated method, and after 48 hours, protoplasts were collected and DNA was extracted for T7EI and PCR-RE assay.

Wheat protoplast preparation and transformation

1) Taking young and tender leaves of wheat, cutting the middle part of the young and tender leaves into 0.5-1mm threads, putting the young and tender leaves into 0.6M Mannitol solution for 10 minutes in the dark, filtering the leaves by using a filter screen, putting the leaves into 50ml of enzyme solution for digestion at 20-25 ℃ in the dark by slowly shaking for 5 hours at 10 rmp.

2) The enzymatic product was diluted with 10ml of W5 and filtered through a 75 μm nylon filter in a round-bottom centrifuge tube (50 ml).

3) Centrifuge at 23 deg.C for 3min at 100g, and discard the supernatant.

4) The suspension was gently suspended with W5 ml, and the protoplasts were gradually settled by placing on ice for 30min, and the supernatant was discarded.

5) An appropriate amount of MMG was added to suspend and put on ice for transformation.

6) 10-20. Mu.g of plasmid, 200. Mu.l of protoplast (approx. 4X 10) were added to a 2ml centrifuge tube ⁵ Cell), 220 μ l of newly prepared PEG solution, mixing, and standing in dark at room temperature for 10-20 min to induce transformationAnd (4) transforming.

7) After the induction transformation, slowly adding 880. Mu.l of W5 solution, slightly inverting and mixing, horizontally centrifuging at 100g for 3min, and absorbing and discarding the supernatant.

8) Add 2ml W5 solution for heavy suspension, transfer to six-well plate, room temperature (or 25 degrees C) dark culture. For extracting protoplast genome DNA, 48h of culture is required.

Preparation and transformation of rice protoplasts:

1) The seedling leaf sheath section was selected to isolate protoplasts and cut with a sharp blade to approximately 0.5mm width.

2) Immediately after the incision, the cells were transferred to 0.6M Mannitol solution and left to stand for 10min in the dark.

3) The Mannitol solution was filtered off, transferred to the enzymatic hydrolysate, and evacuated for 30min in the dark.

4) Enzymolysis for 5-6h in the dark while slowly shaking (decolorization shaker, speed 10).

5) After the enzymatic hydrolysis was completed, an equal volume of W5 was added and the protoplasts were released by shaking horizontally for 10 sec.

6) Protoplasts were filtered through a 40 μm nylon membrane into a 50ml round-bottom centrifuge tube and rinsed with W5 solution.

7) 250g of the suspension was horizontally centrifuged for 3min to precipitate protoplasts, and the supernatant was aspirated and discarded.

8) Add 10ml W5 to resuspend the protoplast, centrifuge at 250g for 3min, discard the supernatant.

9) Adding proper amount of MMG solution to resuspend protoplast at a concentration of 2X 10 ⁶ /ml。

Note: all the above steps were carried out at room temperature.

10 2ml centrifuge tube with 10-20. Mu.g plasmid, 200. Mu.l protoplast (approximately 4X 10) ⁵ Cells), 220 mul of newly prepared PEG solution, mixing, and placing in the dark at room temperature for 10-20 minutes to induce transformation.

11 880. Mu.l of W5 solution was slowly added after the induction transformation was completed, the mixture was gently inverted and mixed, 250g was horizontally centrifuged for 3min, and the supernatant was aspirated.

12 2ml of WI solution, transferred to a six-well plate, and cultured at room temperature (or 25 ℃) in the dark for 48 hours if used for extracting protoplast genomic DNA.

PCR/RE detection:

1) And extracting plant genome DNA.

2) Synthesizing gene specific primer, amplifying the segment containing target site, the length is 350-1000 bp:

10×EasyTaq Buffer	5μl
		dNTP(2.5mM)	4μl
forward primer (10. Mu.M)	2μl
		Forward primer (10. Mu.M)	2μl
Easy Taq	0.5μl
		DNA	2μl
ddH 2 O	To 50. Mu.l

3) The general reaction conditions are: denaturation at 94 deg.C for 5min; carrying out denaturation at 94 ℃ for 30s, renaturation at 58 ℃ for 30s, extension at 72 ℃ for 30s, and amplification for 30-35 cycles; keeping the temperature at 72 ℃ for 5min; keeping the temperature at 12 ℃.5 μ l of PCR product was electrophoretically detected.

4) The PCR product is digested by restriction enzymes, and the general digestion system is as follows:

10×Fastdigest Buffer	2μl
		restriction enzyme	1μl
PCR product	3-5μl
		ddH 2 O	To 20. Mu.l

5) And carrying out enzyme digestion for 2-3h at 37 ℃. Detection by 1.2% agarose gel electrophoresis.

6) The non-cut mutation band in the purified PCR product was recovered and TA cloned. The reaction system is as follows:

pEasy-T Vector	1μl
		recovered uncut PCR product	4μl

7) Connecting for 10min at 22 ℃, transforming E.coli competent cells, coating LB solid plates (Amp 100, IPTG and X-gal), culturing for 12-16h, selecting white colonies, identifying positive clones, and sequencing.

Deep sequencing

And (3) converting different sgRNA expression vectors and the Ubi-CPF1-PBE/ABE expression vector into wheat and rice plastids for 48 hours, collecting protoplasts, and extracting DNA for deep sequencing. In the first round of PCR, the target region is amplified using site-specific primers. In two rounds of PCR, forward and reverse tags were added to the ends of the PCR products for library construction. Equal amounts of different PCR products were pooled. The samples were then sequenced at the Beijing Genomics Institute using Illumina High-Seq 4000.

Examples

Example 1 CPF1-mediated optimization of plant genome cleavage activity.

The editing activity of CPF1 in plant cells varies greatly among different articles, and the cleavage activity between different types of CPF1 also varies greatly.

In the embodiment, the nucleous state of AsCPF1, fnCPF1 and LbCPF1 is optimized, and the promoter of crRNA is optimized, so that the cutting activity of CPF1 in plant cells is improved. Vectors for 1-4 NLS of AsCPF1, fnCPF1 and LbCPF1 were constructed, as well as different vectors for U3/U6 and UBI-initiated production of crRNA by ribozymes (see FIG. 1). As can be seen from the results of PCR/RE, three CPF1 of 2NLS can work, and the LbCPF1 has high efficiency (the corresponding amino acid sequences can be easily obtained by using the coding sequences of ASCPF1-2NLS, FNCPF1-2NLS and LBCPF1-2NLS as SEQ ID NOS: 5-7, respectively). Aiming at the target site of the OsPDS gene, the efficiency of 2NLS-LbCPF1 is higher than that of NLS-LbCPF1 and higher than that of other reported constructs.

Example 2 CPF1-mediated mutation of plant genome C into T (CPF 1-PBE)

Referring to the characteristics of CPF1 in the plant cell cleavage activity, the following dCPF1-PBE system was constructed: dAsCPF1-2NLS-PBE, dFnCPF1-2NLS-PBE, dLbCPF1-2NLS-PBE. The NLS at the C end is respectively placed at one end of the UGI and at two ends of the UGI. crRNA is initiated with UBIl and cleaved with ribozymes. The editing activity was detected by using PCR/RE test results, and it was shown that the editing activity was detected by dFnCPf1 and dLbCPF1, and the activity of NLS at one end of UGI was high (SEQ ID NO:8,9 shows the coding sequences of dFNCPF1-PBE-2NLS and dLbCPF1-2NLS-PBE, respectively, and the corresponding amino acid sequences were easily obtained). In addition, a boost of dCPF1-PBE2-X was constructed, i.e., an intron was added after the promoter ZmUbi-1 to increase the expression of dCPF1-PBE (SEQ ID NO:10 shows dLBCPF1-PBE-2NLS expression cassette comprising the promoter ZmUbi-1 and intron sequences).

Example 3 CPF1-mediated mutation of plant genomes A to G (CPF 1-ABE)

The following CPF1-ABE system was constructed: dAsCPF1-1NLS-ABE, dFnCPF1-NLS-ABE, dLbCPF1-1NLS-ABE, and dAsCPF1-2NLS-ABE, dFnCPF1-2NLS-ABE, dLbCPF1-2NLS-ABE, which ABE again includes ABE7.9 and ABE7.10 two versions. crRNA is initiated with UBIl and cleaved with ribozymes.

The results using the GFP base editing reporter system of fig. 3E show that: both dFnCPF1-ABE7.10 (SEQ ID NO: 11) and dLbCPF1-ABE7.9 and dLbCPF1-ABE7.10 (SEQ ID NO: 12) work, with 7.10 being higher than 7.9 (FIG. 3F).

The PCR/RE detection result shows that the activity of the dLbCPF1-ABE7.10 is detected, and the 2NLS is higher than 1NLS. In addition, two enhanced versions of dCPF1-ABE2 were constructed, i.e., an intron was added after the UBI1 promoter to increase the expression of dCPF1-ABE (dCPF 1-ABE 2-X1) (SEQ ID NO. 13), and ABE was constructed at the C-terminus of CPF1 (dCPF 1-ABE 2-X2/X3) (SEQ ID NO.14, 15), and the results of the reporter system were compiled using the GFP base of FIG. 3E: the editing activity of dCPF1-ABE2-X2/X3 is higher than that of dLbCPF1-ABE7.10 (FIG. 3G).

Example 4 CPF1-mediated optimization of Gene editing

In order to improve the editing efficiency of CPF1, the CPF1 system is optimized, and all expression vectors for CPF 1-mediated editing are started by BdUbi10 promoter to increase the expression quantity. Furthermore, the coding efficiency of CPF1 was improved by increasing the expression of mRNA by promoting the promoter of crRNA using a type II promoter and inserting crRNA Array into the 5'UTR or 3' UTR region of the expressed gene.

Description of the related sequences:

SEQ ID NO.1 cytidine deaminase amino acid sequence

SEQ ID No.2 uracil DNA glycosylase inhibitor (UGI) amino acid sequence

SEQ ID NO.3 wild type ecTadA amino acid sequence

SEQ ID NO.4 ecTadA-derived DNA-dependent adenine deaminase (ABE version 7.10) amino acid sequence

SEQ ID NO.5 ASCPF1-2NLS coding sequence

SEQ ID NO.6 FNCPF1-2NLS coding sequence

SEQ ID NO.7 LBCPF1-2NLS coding sequence

SEQ ID NO.8 dFNCPF1-PBE-2NLS coding sequence

SEQ ID NO.9 dLBCPF1-PBE-2NLS coding sequence

SEQ ID NO.10 promoter + intron + dLBCPF1-PBE-2NLS coding sequence

SEQ ID NO.11 dFNCPF1-ABE7.10-2NLS coding sequence

SEQ ID NO.12 dLBCPF1-ABE7.10-2NLS coding sequence

SEQ ID NO.13 promoter + Intron + dLBCPF1-ABE2-X coding sequence

SEQ ID NO.14 LBCPF1-ABE2-X2 coding sequence

SEQ ID NO.15 LBCPF1-ABE2-X3 coding sequence

SEQ ID NO.16 PJIT163-GFP

SEQ ID NO:17 pBUI-mGFP

18 ASCPF1 amino acid sequence of SEQ ID NO

19 FNCPF1 amino acid sequence of SEQ ID NO

20 LBCPF1 amino acid sequence of SEQ ID NO

21 ASCPF1-2NLS amino acid sequence of SEQ ID NO

SEQ ID NO 22 FNCPF1-2NLS amino acid sequence

23 LBCPF1-2NLS amino acid sequence of SEQ ID NO

24 dFNCPF1-PBE-2NLS amino acid sequence of SEQ ID NO

25 dLBCPF1-PBE-2NLS amino acid sequence of SEQ ID NO

SEQ ID NO 26 dFNCPF1-ABE7.10-2NLS amino acid sequence

27 dLBCPF1-ABE7.10-2NLS amino acid sequence of SEQ ID NO

28 LBCPF1-ABE2-X2 amino acid sequence of SEQ ID NO

29 LBCPF1-ABE2-X3 amino acid sequence of SEQ ID NO

30 promoter + Intron nucleotide sequence of SEQ ID NO

Sequence listing

<110> institute of genetics and developmental biology of Chinese academy of sciences

<120> CPF1 protein-based base editing system and method

<130> 149132

<150> 201711403490.X

<151> 2017-12-22

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atgacgcagt tcgaggggtt caccaacctc taccaggtca gcaagacgct gcggttcgag 60

ctgattccgc agggcaagac cctcaagcac atccaggagc agggctttat cgaggaggac 120

aaagcgcgga acgaccacta caaggagctc aagccgatca tcgaccggat ctacaagacc 180

tacgcggacc agtgcctgca gctcgtgcag ctcgactggg agaacctctc ggccgccatc 240

gactcctacc gcaaggagaa gacggaggag acacgcaacg cgctcatcga agaacaggct 300

acgtatcgca acgctatcca cgactacttc atcgggcgca cagataatct caccgacgcc 360

atcaacaagc gccacgccga aatctataag gggctgttta aggccgagct gttcaatggc 420

aaagtgctga agcaactggg caccgtcacg accacagagc atgagaacgc cctcctccgg 480

tcgttcgaca agtttacgac atactttagc ggcttttacg agaaccgcaa aaacgtgttc 540

agcgccgagg acattagcac cgccatcccg cataggatcg tgcaagacaa cttcccgaag 600

ttcaaggaga actgccacat cttcacccgc ctcatcacgg ccgtgccgtc cctccgcgag 660

cacttcgaga acgtgaagaa ggccatcggg atcttcgtgt ccacctcgat cgaagaggtg 720

ttctccttcc ccttctacaa tcagctgctg acgcagaccc agatcgacct ctacaaccag 780

ctcctcggcg gcatctcccg cgaggccggc accgagaaga tcaagggcct gaacgaggtg 840

ctcaatctcg ccatccagaa gaacgacgaa accgcgcaca tcattgcctc actcccacat 900

aggtttatcc ccctgtttaa gcagatcctc tccgaccgca acacgttgtc cttcatcctc 960

gaggagttca agagcgacga ggaggtcatc cagtccttct gcaagtacaa gaccctcctc 1020

cgcaacgaga atgtgctcga aaccgccgag gcgctgttca atgagctcaa tagcatcgat 1080

ctcacccaca tcttcatctc ccataagaag ctggaaacca tctccagcgc cctgtgcgac 1140

cactgggaca ccctccgcaa cgccctctac gagcggcgca tcagcgagct caccggcaag 1200

atcacgaagt cggcgaaaga gaaagtgcaa aggagcctca agcacgagga cattaacctg 1260

caggagatca tctccgccgc gggcaaggag ctgtccgagg ctttcaagca gaagacctcg 1320

gagatcctct ctcacgccca cgcggccctg gaccagccgc tgccgacgac cctgaaaaag 1380

caagaggaaa aggaaatcct caagtcccag ctggacagcc tcctggggct gtaccacctt 1440

ctcgactggt tcgccgtgga cgagtccaac gaggtcgatc cggagtttag cgcccgcctc 1500

accgggatca agcttgagat ggaacctagc ttgagcttct ataataaggc gcgcaattac 1560

gcgaccaaga agccgtattc cgtggagaag ttcaagctga acttccaaat gcccaccctg 1620

gccagcgggt gggacgttaa caaagagaag aacaacggag ccattctttt cgtgaaaaat 1680

gggttgtatt atttgggaat tatgccgaaa caaaaaggca ggtacaaggc gctcagcttc 1740

gagccaactg agaagacctc cgagggcttc gacaagatgt actacgatta ttttcctgac 1800

gctgcaaaga tgataccgaa gtgcagcact cagcttaagg cggtgacggc gcactttcag 1860

acccatacca cccccatcct cctctccaac aacttcatcg agccgctcga gatcaccaag 1920

gagatatacg atctgaataa tccagaaaag gaacccaaga agttccagac cgcctacgcc 1980

aagaagacgg gcgatcaaaa ggggtataga gaggcgctct gcaagtggat cgacttcacg 2040

cgcgatttcc tcagcaagta caccaagaca acctccatcg atctctcttc cctccgcccc 2100

tcttcccagt acaaggacct cggggagtac tacgccgaac tcaacccact cctgtatcac 2160

atctcgtttc agcgtatcgc ggaaaaggag atcatggacg ccgtcgaaac cggcaagttg 2220

tatcttttcc aaatctataa caaggacttc gcgaagggcc accacgggaa gccaaacctg 2280

cacaccctct actggacagg cctcttcagc ccggaaaatc tcgcgaagac gagcataaag 2340

ctgaacggcc aggcagaact cttctacagg ccgaagtcca ggatgaagcg catggctcat 2400

cgcctcggtg agaagatgct gaacaagaag ctgaaagatc aaaagacgcc aatccctgat 2460

acactgtatc aggagctgta cgattacgtg aaccaccgcc tctcacacga cctcagcgac 2520

gaggcccgcg cgctcctgcc aaacgtcatc acgaaggagg tcagccacga gatcataaag 2580

gatcggcggt ttacctctga caagttcttt ttccatgtcc ccatcacgct gaactaccag 2640

gccgcgaata gcccgtccaa gttcaaccag cgggtcaacg cgtatctcaa ggagcaccca 2700

gagacaccca taatcgggat tgaccggggg gagcggaacc tcatctacat caccgtcatc 2760

gactccaccg gaaagattct cgagcaacgg tcgctcaata ccatccagca gttcgactac 2820

cagaagaagc tcgacaaccg ggagaaggaa cgcgtcgccg cgaggcaggc ctggtccgta 2880

gtgggcacga tcaaagacct gaagcagggc tatctcagcc aggtcatcca tgagatagtg 2940

gatctcatga tccactacca agccgtcgtg gtcctcgaga atctcaattt cggattcaaa 3000

tccaagcgca caggcatcgc cgagaaggcg gtgtaccaac agttcgagaa aatgcttatc 3060

gacaagctca attgcctggt gctcaaggac tatccggcgg agaaggtcgg gggggtcctc 3120

aatccgtatc agctgaccga ccagtttacg tcatttgcga agatgggcac ccagagcggc 3180

ttccttttct atgtcccggc cccatatacc tcaaagattg atcccttgac cggattcgtg 3240

gacccgtttg tctggaagac catcaagaac catgagtcgc gtaagcattt cctggagggt 3300

ttcgacttcc tgcactatga tgtaaaaacc ggagacttca tcctgcattt caagatgaat 3360

cggaacctct ccttccagcg gggactccct ggcttcatgc ccgcttggga tatcgttttt 3420

gagaaaaatg aaacccaatt cgacgccaaa ggcacgcctt tcatcgcggg caagaggatt 3480

gtccctgtaa ttgagaacca tagattcacc gggcgttacc gtgacctgta ccccgcaaac 3540

gaactcatcg ccctcctgga ggagaaaggc atcgttttcc gcgacgggtc aaatatcctc 3600

cccaaactgc tcgagaacga tgatagccac gctattgaca cgatggtagc gctcatcaga 3660

tccgtgctgc aaatgagaaa ttcaaatgct gccactgggg aggattacat caactcccct 3720

gtgcgtgatc tcaatggcgt gtgcttcgat tctagatttc agaatcctga gtggccgatg 3780

gatgccgatg ctaacggcgc ataccacata gcattgaaag gacaactgtt gttgaaccat 3840

ctcaaggaga gcaaggacct taagctgcag aacggcatca gcaaccagga ttggcttgcc 3900

tatatccaag agctccgcaa ttccggcggc agcccaaaga agaagaggaa ggtgagcggc 3960

ggcagcccaa agaagaagcg caaggtctag 3990

<210> 6

<211> 3969

<212> DNA

<213> Artificial Sequence

<220>

<223> FNCPF1-2NLS

<400> 6

atgtccatct accaggagtt cgtcaataag tactcactct ctaagaccct gcggttcgag 60

ctgatcccgc agggcaagac actcgagaac atcaaggcgc gcggcctgat tctcgacgat 120

gagaagcggg ccaaggacta caagaaggcg aagcagatca ttgataagta ccaccagttc 180

ttcatcgagg agattctgtc cagcgtgtgc atctctgagg atctcctgca gaattactcc 240

gacgtctact tcaagctcaa gaagtctgac gatgacaacc tgcagaagga tttcaagtcc 300

gccaaggaca ccatcaagaa gcagatttct gagtacatca aggattccga gaagttcaag 360

aatctcttca accagaatct gattgatgcg aagaagggcc aggagtctga cctgatcctc 420

tggctgaagc agtccaagga caatggcatt gagctgttca aggccaacag cgatatcacc 480

gatattgacg aggcgctgga gatcattaag tcattcaagg gctggaccac atacttcaag 540

ggcttccatg agaaccggaa gaatgtgtac tcatctaacg acattccgac ctccatcatc 600

tacaggatcg tcgatgacaa tctgccaaag ttcctcgaga acaaggccaa gtacgagtcc 660

ctcaaggaca aggccccgga ggcgattaat tacgagcaga tcaagaagga tctggcggag 720

gagctgacct tcgatatcga ctacaagaca agcgaggtga accagagggt gttctccctc 780

gatgaggtgt tcgagatcgc caatttcaac aattacctga accagtccgg cattaccaag 840

ttcaatacaa tcattggcgg caagttcgtc aacggcgaga ataccaagcg caagggcatt 900

aacgagtaca tcaatctcta ctcccagcag atcaacgaca agaccctgaa gaagtacaag 960

atgtctgtgc tcttcaagca gatcctgtcc gatacagagt ccaagagctt cgtcattgat 1020

aagctcgagg acgacagcga cgtggtcacc acaatgcagt cattctacga gcagatcgcc 1080

gcgttcaaga ccgtggagga gaagagcatt aaggagacac tctcactcct gttcgatgac 1140

ctgaaggccc agaagctcga cctgagcaag atctacttca agaacgataa gagcctcaca 1200

gacctgtcac agcaggtgtt cgatgactac tcagtgattg gcaccgccgt cctcgagtac 1260

attacacagc agatcgcgcc aaagaacctc gataatcctt ctaagaagga gcaggagctg 1320

atcgccaaga aaaccgagaa ggcgaagtac ctctccctgg agacaattaa gctcgccctg 1380

gaggagttca ataagcacag ggatattgac aagcagtgcc gcttcgagga gatcctcgcg 1440

aacttcgccg cgatcccaat gattttcgat gagatcgccc agaacaagga caatctggcg 1500

cagatctcta ttaagtacca gaaccagggc aagaaggacc tcctgcaggc ctccgcagag 1560

gacgacgtga aggccatcaa ggatctcctg gaccagacca acaatctcct gcacaagctc 1620

aagatcttcc atatttcaca gtctgaggat aaggccaata tcctcgataa ggacgagcat 1680

ttctacctgg tgttcgagga gtgctacttc gagctggcga acattgtccc tctgtacaac 1740

aagattagga attacatcac acagaagccg tacagcgacg agaagttcaa gctcaacttc 1800

gagaattcaa ccctggccaa cggctgggat aagaataagg agcctgacaa cacagcgatc 1860

ctcttcatca aggacgacaa gtactacctg ggcgtgatga ataagaagaa caataagatc 1920

ttcgatgaca aggccattaa ggagaacaag ggcgagggct acaagaagat cgtgtacaag 1980

ctcctgcctg gcgccaataa gatgctcccg aaggtgttct tctccgcgaa gtccattaag 2040

ttctacaacc caagcgagga tatcctcagg atcaggaacc actctaccca tacaaagaac 2100

ggctcccctc agaagggcta cgagaagttc gagttcaata tcgaggattg ccggaagttc 2160

attgacttct acaagcagtc catcagcaag caccctgagt ggaaggattt cggcttccgc 2220

ttcagcgaca cccagcggta caactcaatc gatgagttct acagggaggt ggagaatcag 2280

ggctacaagc tcacattcga gaacatttca gagtcttaca tcgactccgt ggtcaatcag 2340

ggcaagctct acctgttcca gatctacaac aaggatttca gcgcctactc aaagggcagg 2400

ccgaacctcc ataccctgta ctggaaggcg ctcttcgatg agcgcaatct gcaggacgtg 2460

gtctacaagc tcaacggcga ggccgagctg ttctaccgca agcagtctat tccgaagaag 2520

atcacacacc cagcgaagga ggccatcgcg aacaagaata aggacaatcc gaagaaggag 2580

tccgtgttcg agtacgatct cattaaggac aagcggttca ccgaggataa gttcttcttc 2640

cattgcccaa tcacaattaa cttcaagtcc agcggcgcca acaagttcaa tgacgagatc 2700

aatctcctgc tcaaggagaa ggcgaacgat gtgcatatcc tctccattga ccgcggcgag 2760

cggcatctcg cctactacac cctggtcgat ggcaagggca atatcattaa gcaggatacc 2820

ttcaacatca ttggcaatga ccgcatgaaa accaactacc acgataagct cgccgcgatc 2880

gagaaggata gggacagcgc gcgcaaggac tggaagaaga tcaacaatat taaggagatg 2940

aaggagggct acctctcaca ggtggtccat gagattgcca agctcgtgat cgagtacaat 3000

gcgattgtgg tgttcgagga cctcaacttc ggcttcaagc ggggcaggtt caaggtggag 3060

aagcaggtct accagaagct ggagaagatg ctcatcgaga agctcaatta cctggtgttc 3120

aaggataacg agttcgacaa gaccggagga gtcctcaggg cataccagct gaccgcgcca 3180

ttcgagacat tcaagaagat gggcaagcag acaggcatca tctactacgt gccagccggc 3240

ttcacctcca agatctgccc tgtgacaggc ttcgtcaacc agctctaccc taagtacgag 3300

tccgtcagca agtcacagga gttcttcagc aagttcgata agatttgcta caatctggac 3360

aagggctact tcgagttctc attcgattac aagaacttcg gcgacaaggc cgcgaagggc 3420

aagtggacca ttgcgtcttt cggctcccgg ctcatcaact tcaggaatag cgataagaac 3480

cacaattggg acaccaggga ggtgtacccg acaaaggagc tggagaagct gctcaaggac 3540

tactcaatcg agtacggcca tggcgagtgc attaaggccg cgatctgcgg cgagagcgat 3600

aagaagttct tcgccaagct gacctcagtc ctcaatacaa tcctgcagat gaggaactct 3660

aagaccggca cagagctgga ctacctgatt tccccagtgg ccgatgtcaa cggcaatttc 3720

ttcgacagcc gccaggcgcc gaagaatatg ccacaggacg ccgacgccaa cggcgcatac 3780

cacatcggcc tcaagggcct gatgctgctc ggacgcatta agaacaatca ggagggcaag 3840

aagctcaacc tcgtgatcaa gaacgaggag tacttcgagt tcgtccaaaa ccggaacaat 3900

tccggcggca gcccaaagaa gaagaggaag gtgagcggcg gcagcccaaa gaagaagcgc 3960

aaggtctag 3969

<210> 7

<211> 3753

<212> DNA

<213> Artificial Sequence

<220>

<223> LBCPF1-2NLS

<400> 7

atgtcaaagc tcgagaaatt caccaactgt tattcgttga gcaaaacact gcggtttaaa 60

gcgattccag tcggcaagac tcaagagaat atagacaata agcggctgtt ggtggaagat 120

gaaaagcgcg cggaagacta caaaggggtg aagaagttgt tggacagata ctacctctct 180

tttatcaatg atgtcttgca ctcaatcaaa ttgaagaatc tgaacaacta catctccctc 240

ttcagaaaga aaacaaggac agaaaaggag aataaggaac ttgaaaattt ggagatcaat 300

ctgaggaaag agatcgcgaa agcctttaaa ggcaacgaag gatacaaaag tctgttcaag 360

aaggatataa ttgagacaat tttgccagag ttcctcgatg acaaggacga gattgcgctg 420

gtcaattcgt tcaacggatt cacaacagca ttcacaggct tctttgataa tcgggaaaat 480

atgttctctg aggaggcaaa gtccacttct attgcgttca ggtgtatcaa tgagaatctc 540

actaggtaca tttccaacat ggatatcttt gagaaggttg acgcaatttt tgacaagcac 600

gaagttcagg agattaagga gaagatcctc aattccgatt atgacgttga ggacttcttc 660

gaaggtgagt tttttaattt cgtgctcact caagagggta tcgacgtgta taatgcgatc 720

atcggtgggt tcgtgactga gtccggtgaa aagattaagg gattgaacga gtatatcaac 780

ctttacaacc aaaagacgaa acagaagctg ccaaagttca agcctcttta caaacaggtt 840

ctttcagacc gcgagtcact ctcgttctat ggggagggct acacttcgga tgaggaagtc 900

ctggaggtgt tcaggaatac tctcaataag aattcggaga ttttctcttc tataaaaaaa 960

ctggaaaagt tgtttaagaa ttttgacgaa tactctagcg ccggcatatt tgtgaaaaac 1020

ggcccggcca tatcaacgat aagtaaagat atcttcggcg aatggaacgt gatcagagac 1080

aaatggaacg cggagtatga cgatattcac ctgaagaaga aggctgtcgt aacggagaag 1140

tacgaggatg atcgcaggaa aagcttcaaa aagatcggaa gtttcagcct ggaacagttg 1200

caggagtatg ctgacgccga tcttagcgtc gtcgagaagt tgaaggagat aatcatccaa 1260

aaggtcgacg agatatataa agtctatgga tcaagtgaaa aactgttcga cgccgacttc 1320

gttttggaga agtccctgaa gaagaacgac gctgttgttg ccattatgaa ggatctgctc 1380

gacagcgtga agagtttcga gaactatatt aaggcttttt tcggggaggg gaaggagact 1440

aacagagatg agtccttcta cggagacttc gtcctcgcgt acgatatact ccttaaggta 1500

gaccacatct acgacgcaat cagaaattac gtgacacaaa agccgtacag caaggacaag 1560

ttcaaactct acttccagaa cccccagttc atgggcggct gggacaagga caaggaaacg 1620

gattacaggg ctacgatcct gaggtatggt tcaaaatact acttggcgat tatggacaag 1680

aagtacgcca agtgtctcca gaagattgac aaagacgatg tcaatggcaa ttatgagaag 1740

atcaactaca agctgcttcc gggtccgaac aagatgctcc caaaggtttt cttcagcaag 1800

aaatggatgg cctactataa cccaagcgag gacatccaga agatttataa gaacggtacg 1860

ttcaagaagg gcgacatgtt caatcttaac gactgtcaca agctgatcga cttcttcaaa 1920

gactcaatta gccggtaccc aaagtggtct aacgcctatg acttcaactt ttcggaaacc 1980

gagaagtaca aggatatagc cggattttat agagaggtgg aagagcaggg ctacaaggtg 2040

tcattcgagt ccgccagcaa gaaggaagtg gacaagctcg tggaagaggg taagctctac 2100

atgttccaga tttataataa agactttagc gataagagcc acgggacacc taatctccac 2160

acaatgtatt tcaagctgct cttcgacgag aataaccacg gccaaatcag gttgtcagga 2220

ggggctgaac tcttcatgcg gcgcgctagc cttaagaagg aggagcttgt agtccaccct 2280

gcgaatagtc caattgcgaa taagaacccg gacaatccta aaaagactac aacattgagc 2340

tacgacgtgt acaaggataa gaggttttcc gaggatcagt acgagctcca catcccgatt 2400

gcgatcaaca agtgcccaaa gaatattttc aagataaaca cagaggtgcg tgtactcctg 2460

aagcatgacg acaatcctta cgtcattggg attgatcggg gcgagaggaa cctcctctat 2520

attgtggtgg tggacgggaa ggggaacata gtcgaacagt actcccttaa cgaaataatt 2580

aacaatttca acggcatccg tatcaagacc gactaccatt cgttgctgga caagaaggag 2640

aaggagagat ttgaggcgcg gcaaaattgg acaagtatcg agaacatcaa ggaactcaaa 2700

gcaggttata tctctcaagt tgtgcataag atatgcgagc tggttgagaa gtatgacgca 2760

gtgatcgctc ttgaggacct caactcgggc tttaagaatt ctagagttaa agtggagaag 2820

caggtctatc aaaagttcga gaagatgctt atagataagc tcaactacat ggtcgataag 2880

aaatcgaacc catgtgccac cggcggcgca ctcaaaggtt accaaataac aaacaaattc 2940

gagtccttca aatcgatgag tactcagaat gggttcatat tttatatacc ggcgtggctt 3000

acgtctaaga tcgacccgtc aactggtttt gtcaacctgt tgaagacgaa atacacgtcc 3060

attgccgatt cgaaaaagtt catatctagt tttgatcgta ttatgtacgt cccagaggaa 3120

gatcttttcg agtttgctct cgactacaaa aacttttcgc ggaccgatgc ggattacatt 3180

aaaaaatgga aactctattc gtacggcaac agaatcagga tttttcgcaa ccctaagaag 3240

aataacgtct ttgattggga ggaagtttgc ttgactagcg cgtacaagga gctctttaat 3300

aagtatggca ttaactacca acagggtgat atcagagcac tgctttgcga acaatctgac 3360

aaggctttct actcatcctt catggctttg atgagcctga tgctccagat gagaaattca 3420

attacaggca gaaccgacgt ggatttcttg atctccccgg ttaaaaattc tgatggcatc 3480

ttttacgata gcaggaacta tgaagcgcaa gagaatgcga ttctgccaaa aaatgcagac 3540

gccaacggtg cctataacat cgccaggaaa gtcctgtggg cgatcggcca gttcaaaaag 3600

gccgaagacg aaaaattgga caaggtcaaa atcgctatca gcaacaaaga gtggctggag 3660

tatgctcaga catccgtaaa gcattccggc ggcagcccaa agaagaagag gaaggtgagc 3720

ggcggcagcc caaagaagaa gcgcaaggtc tag 3753

<210> 8

<211> 4995

<212> DNA

<213> Artificial Sequence

<220>

<223> dFNCPF1-PBE-2NLS

<400> 8

atgccaaaga agaagaggaa ggtttcatcg gagaccggcc ctgttgctgt tgaccccacc 60

ctgcggcgga gaatcgagcc acacgagttc gaggtgttct tcgacccaag ggagctccgc 120

aaggagacgt gcctcctgta cgagatcaac tggggcggca ggcactccat ctggaggcac 180

accagccaaa acaccaacaa gcacgtggag gtcaacttca tcgagaagtt caccaccgag 240

aggtacttct gcccaaacac ccgctgctcc atcacctggt tcctgtcctg gagcccatgc 300

ggcgagtgct ccagggccat caccgagttc ctcagccgct acccacacgt caccctgttc 360

atctacatcg ccaggctcta ccaccacgcc gacccaagga acaggcaggg cctccgcgac 420

ctgatctcca gcggcgtgac catccaaatc atgaccgagc aggagtccgg ctactgctgg 480

aggaacttcg tcaactactc cccaagcaac gaggcccact ggccaaggta cccacacctc 540

tgggtgcgcc tctacgtgct cgagctgtac tgcatcatcc tcggcctgcc accatgcctc 600

aacatcctga ggcgcaagca accacagctg accttcttca ccatcgccct ccaaagctgc 660

cactaccaga ggctcccacc acacatcctg tgggctaccg gcctcaagtc cggcagcgag 720

acgccaggca cctccgagag cgctacgcct gaacttaagt ccatctacca ggagttcgtc 780

aataagtact cactctctaa gaccctgcgg ttcgagctga tcccgcaggg caagacactc 840

gagaacatca aggcgcgcgg cctgattctc gacgatgaga agcgggccaa ggactacaag 900

aaggcgaagc agatcattga taagtaccac cagttcttca tcgaggagat tctgtccagc 960

gtgtgcatct ctgaggatct cctgcagaat tactccgacg tctacttcaa gctcaagaag 1020

tctgacgatg acaacctgca gaaggatttc aagtccgcca aggacaccat caagaagcag 1080

atttctgagt acatcaagga ttccgagaag ttcaagaatc tcttcaacca gaatctgatt 1140

gatgcgaaga agggccagga gtctgacctg atcctctggc tgaagcagtc caaggacaat 1200

ggcattgagc tgttcaaggc caacagcgat atcaccgata ttgacgaggc gctggagatc 1260

attaagtcat tcaagggctg gaccacatac ttcaagggct tccatgagaa ccggaagaat 1320

gtgtactcat ctaacgacat tccgacctcc atcatctaca ggatcgtcga tgacaatctg 1380

ccaaagttcc tcgagaacaa ggccaagtac gagtccctca aggacaaggc cccggaggcg 1440

attaattacg agcagatcaa gaaggatctg gcggaggagc tgaccttcga tatcgactac 1500

aagacaagcg aggtgaacca gagggtgttc tccctcgatg aggtgttcga gatcgccaat 1560

ttcaacaatt acctgaacca gtccggcatt accaagttca atacaatcat tggcggcaag 1620

ttcgtcaacg gcgagaatac caagcgcaag ggcattaacg agtacatcaa tctctactcc 1680

cagcagatca acgacaagac cctgaagaag tacaagatgt ctgtgctctt caagcagatc 1740

ctgtccgata cagagtccaa gagcttcgtc attgataagc tcgaggacga cagcgacgtg 1800

gtcaccacaa tgcagtcatt ctacgagcag atcgccgcgt tcaagaccgt ggaggagaag 1860

agcattaagg agacactctc actcctgttc gatgacctga aggcccagaa gctcgacctg 1920

agcaagatct acttcaagaa cgataagagc ctcacagacc tgtcacagca ggtgttcgat 1980

gactactcag tgattggcac cgccgtcctc gagtacatta cacagcagat cgcgccaaag 2040

aacctcgata atccttctaa gaaggagcag gagctgatcg ccaagaaaac cgagaaggcg 2100

aagtacctct ccctggagac aattaagctc gccctggagg agttcaataa gcacagggat 2160

attgacaagc agtgccgctt cgaggagatc ctcgcgaact tcgccgcgat cccaatgatt 2220

ttcgatgaga tcgcccagaa caaggacaat ctggcgcaga tctctattaa gtaccagaac 2280

cagggcaaga aggacctcct gcaggcctcc gcagaggacg acgtgaaggc catcaaggat 2340

ctcctggacc agaccaacaa tctcctgcac aagctcaaga tcttccatat ttcacagtct 2400

gaggataagg ccaatatcct cgataaggac gagcatttct acctggtgtt cgaggagtgc 2460

tacttcgagc tggcgaacat tgtccctctg tacaacaaga ttaggaatta catcacacag 2520

aagccgtaca gcgacgagaa gttcaagctc aacttcgaga attcaaccct ggccaacggc 2580

tgggataaga ataaggagcc tgacaacaca gcgatcctct tcatcaagga cgacaagtac 2640

tacctgggcg tgatgaataa gaagaacaat aagatcttcg atgacaaggc cattaaggag 2700

aacaagggcg agggctacaa gaagatcgtg tacaagctcc tgcctggcgc caataagatg 2760

ctcccgaagg tgttcttctc cgcgaagtcc attaagttct acaacccaag cgaggatatc 2820

ctcaggatca ggaaccactc tacccataca aagaacggct cccctcagaa gggctacgag 2880

aagttcgagt tcaatatcga ggattgccgg aagttcattg acttctacaa gcagtccatc 2940

agcaagcacc ctgagtggaa ggatttcggc ttccgcttca gcgacaccca gcggtacaac 3000

tcaatcgatg agttctacag ggaggtggag aatcagggct acaagctcac attcgagaac 3060

atttcagagt cttacatcga ctccgtggtc aatcagggca agctctacct gttccagatc 3120

tacaacaagg atttcagcgc ctactcaaag ggcaggccga acctccatac cctgtactgg 3180

aaggcgctct tcgatgagcg caatctgcag gacgtggtct acaagctcaa cggcgaggcc 3240

gagctgttct accgcaagca gtctattccg aagaagatca cacacccagc gaaggaggcc 3300

atcgcgaaca agaataagga caatccgaag aaggagtccg tgttcgagta cgatctcatt 3360

aaggacaagc ggttcaccga ggataagttc ttcttccatt gcccaatcac aattaacttc 3420

aagtccagcg gcgccaacaa gttcaatgac gagatcaatc tcctgctcaa ggagaaggcg 3480

aacgatgtgc atatcctctc cattgcccgc ggcgagcggc atctcgccta ctacaccctg 3540

gtcgatggca agggcaatat cattaagcag gataccttca acatcattgg caatgaccgc 3600

atgaaaacca actaccacga taagctcgcc gcgatcgaga aggataggga cagcgcgcgc 3660

aaggactgga agaagatcaa caatattaag gagatgaagg agggctacct ctcacaggtg 3720

gtccatgaga ttgccaagct cgtgatcgag tacaatgcga ttgtggtgtt cgaggacctc 3780

aacttcggct tcaagcgggg caggttcaag gtggagaagc aggtctacca gaagctggag 3840

aagatgctca tcgagaagct caattacctg gtgttcaagg ataacgagtt cgacaagacc 3900

ggaggagtcc tcagggcata ccagctgacc gcgccattcg agacattcaa gaagatgggc 3960

aagcagacag gcatcatcta ctacgtgcca gccggcttca cctccaagat ctgccctgtg 4020

acaggcttcg tcaaccagct ctaccctaag tacgagtccg tcagcaagtc acaggagttc 4080

ttcagcaagt tcgataagat ttgctacaat ctggacaagg gctacttcga gttctcattc 4140

gattacaaga acttcggcga caaggccgcg aagggcaagt ggaccattgc gtctttcggc 4200

tcccggctca tcaacttcag gaatagcgat aagaaccaca attgggacac cagggaggtg 4260

tacccgacaa aggagctgga gaagctgctc aaggactact caatcgagta cggccatggc 4320

gagtgcatta aggccgcgat ctgcggcgag agcgataaga agttcttcgc caagctgacc 4380

tcagtcctca atacaatcct gcagatgagg aactctaaga ccggcacaga gctggactac 4440

ctgatttccc cagtggccga tgtcaacggc aatttcttcg acagccgcca ggcgccgaag 4500

aatatgccac aggacgccga cgccaacggc gcataccaca tcggcctcaa gggcctgatg 4560

ctgctcggac gcattaagaa caatcaggag ggcaagaagc tcaacctcgt gatcaagaac 4620

gaggagtact tcgagttcgt ccaaaaccgg aacaatacgc gtgactccgg cggcagcacc 4680

aacctgtccg acatcatcga gaaggagacg ggcaagcaac tcgtgatcca ggagagcatc 4740

ctcatgctgc cagaggaggt ggaggaggtc atcggcaaca agccagagtc cgacatcctg 4800

gtgcacaccg cctacgacga gtccaccgac gagaacgtca tgctcctgac cagcgacgcc 4860

ccagagtaca agccatgggc cctcgtcatc caggacagca acggggagaa caagatcaag 4920

atgctgtcgg gggggagccc aaagaagaag cggaaggtga gcggcggcag cccaaagaag 4980

aagcgcaagg tctag 4995

<210> 9

<211> 4779

<212> DNA

<213> Artificial Sequence

<220>

<223> dLBCPF1-PBE-2NLS

<400> 9

atgccaaaga agaagaggaa ggtttcatcg gagaccggcc ctgttgctgt tgaccccacc 60

ctgcggcgga gaatcgagcc acacgagttc gaggtgttct tcgacccaag ggagctccgc 120

aaggagacgt gcctcctgta cgagatcaac tggggcggca ggcactccat ctggaggcac 180

accagccaaa acaccaacaa gcacgtggag gtcaacttca tcgagaagtt caccaccgag 240

aggtacttct gcccaaacac ccgctgctcc atcacctggt tcctgtcctg gagcccatgc 300

ggcgagtgct ccagggccat caccgagttc ctcagccgct acccacacgt caccctgttc 360

atctacatcg ccaggctcta ccaccacgcc gacccaagga acaggcaggg cctccgcgac 420

ctgatctcca gcggcgtgac catccaaatc atgaccgagc aggagtccgg ctactgctgg 480

aggaacttcg tcaactactc cccaagcaac gaggcccact ggccaaggta cccacacctc 540

tgggtgcgcc tctacgtgct cgagctgtac tgcatcatcc tcggcctgcc accatgcctc 600

aacatcctga ggcgcaagca accacagctg accttcttca ccatcgccct ccaaagctgc 660

cactaccaga ggctcccacc acacatcctg tgggctaccg gcctcaagtc cggcagcgag 720

acgccaggca cctccgagag cgctacgcct gaacttaagt caaagctcga gaaattcacc 780

aactgttatt cgttgagcaa aacactgcgg tttaaagcga ttccagtcgg caagactcaa 840

gagaatatag acaataagcg gctgttggtg gaagatgaaa agcgcgcgga agactacaaa 900

ggggtgaaga agttgttgga cagatactac ctctctttta tcaatgatgt cttgcactca 960

atcaaattga agaatctgaa caactacatc tccctcttca gaaagaaaac aaggacagaa 1020

aaggagaata aggaacttga aaatttggag atcaatctga ggaaagagat cgcgaaagcc 1080

tttaaaggca acgaaggata caaaagtctg ttcaagaagg atataattga gacaattttg 1140

ccagagttcc tcgatgacaa ggacgagatt gcgctggtca attcgttcaa cggattcaca 1200

acagcattca caggcttctt tgataatcgg gaaaatatgt tctctgagga ggcaaagtcc 1260

acttctattg cgttcaggtg tatcaatgag aatctcacta ggtacatttc caacatggat 1320

atctttgaga aggttgacgc aatttttgac aagcacgaag ttcaggagat taaggagaag 1380

atcctcaatt ccgattatga cgttgaggac ttcttcgaag gtgagttttt taatttcgtg 1440

ctcactcaag agggtatcga cgtgtataat gcgatcatcg gtgggttcgt gactgagtcc 1500

ggtgaaaaga ttaagggatt gaacgagtat atcaaccttt acaaccaaaa gacgaaacag 1560

aagctgccaa agttcaagcc tctttacaaa caggttcttt cagaccgcga gtcactctcg 1620

ttctatgggg agggctacac ttcggatgag gaagtcctgg aggtgttcag gaatactctc 1680

aataagaatt cggagatttt ctcttctata aaaaaactgg aaaagttgtt taagaatttt 1740

gacgaatact ctagcgccgg catatttgtg aaaaacggcc cggccatatc aacgataagt 1800

aaagatatct tcggcgaatg gaacgtgatc agagacaaat ggaacgcgga gtatgacgat 1860

attcacctga agaagaaggc tgtcgtaacg gagaagtacg aggatgatcg caggaaaagc 1920

ttcaaaaaga tcggaagttt cagcctggaa cagttgcagg agtatgctga cgccgatctt 1980

agcgtcgtcg agaagttgaa ggagataatc atccaaaagg tcgacgagat atataaagtc 2040

tatggatcaa gtgaaaaact gttcgacgcc gacttcgttt tggagaagtc cctgaagaag 2100

aacgacgctg ttgttgccat tatgaaggat ctgctcgaca gcgtgaagag tttcgagaac 2160

tatattaagg cttttttcgg ggaggggaag gagactaaca gagatgagtc cttctacgga 2220

gacttcgtcc tcgcgtacga tatactcctt aaggtagacc acatctacga cgcaatcaga 2280

aattacgtga cacaaaagcc gtacagcaag gacaagttca aactctactt ccagaacccc 2340

cagttcatgg gcggctggga caaggacaag gaaacggatt acagggctac gatcctgagg 2400

tatggttcaa aatactactt ggcgattatg gacaagaagt acgccaagtg tctccagaag 2460

attgacaaag acgatgtcaa tggcaattat gagaagatca actacaagct gcttccgggt 2520

ccgaacaaga tgctcccaaa ggttttcttc agcaagaaat ggatggccta ctataaccca 2580

agcgaggaca tccagaagat ttataagaac ggtacgttca agaagggcga catgttcaat 2640

cttaacgact gtcacaagct gatcgacttc ttcaaagact caattagccg gtacccaaag 2700

tggtctaacg cctatgactt caacttttcg gaaaccgaga agtacaagga tatagccgga 2760

ttttatagag aggtggaaga gcagggctac aaggtgtcat tcgagtccgc cagcaagaag 2820

gaagtggaca agctcgtgga agagggtaag ctctacatgt tccagattta taataaagac 2880

tttagcgata agagccacgg gacacctaat ctccacacaa tgtatttcaa gctgctcttc 2940

gacgagaata accacggcca aatcaggttg tcaggagggg ctgaactctt catgcggcgc 3000

gctagcctta agaaggagga gcttgtagtc caccctgcga atagtccaat tgcgaataag 3060

aacccggaca atcctaaaaa gactacaaca ttgagctacg acgtgtacaa ggataagagg 3120

ttttccgagg atcagtacga gctccacatc ccgattgcga tcaacaagtg cccaaagaat 3180

attttcaaga taaacacaga ggtgcgtgta ctcctgaagc atgacgacaa tccttacgtc 3240

attgggattg ctcggggcga gaggaacctc ctctatattg tggtggtgga cgggaagggg 3300

aacatagtcg aacagtactc ccttaacgaa ataattaaca atttcaacgg catccgtatc 3360

aagaccgact accattcgtt gctggacaag aaggagaagg agagatttga ggcgcggcaa 3420

aattggacaa gtatcgagaa catcaaggaa ctcaaagcag gttatatctc tcaagttgtg 3480

cataagatat gcgagctggt tgagaagtat gacgcagtga tcgctcttga ggacctcaac 3540

tcgggcttta agaattctag agttaaagtg gagaagcagg tctatcaaaa gttcgagaag 3600

atgcttatag ataagctcaa ctacatggtc gataagaaat cgaacccatg tgccaccggc 3660

ggcgcactca aaggttacca aataacaaac aaattcgagt ccttcaaatc gatgagtact 3720

cagaatgggt tcatatttta tataccggcg tggcttacgt ctaagatcga cccgtcaact 3780

ggttttgtca acctgttgaa gacgaaatac acgtccattg ccgattcgaa aaagttcata 3840

tctagttttg atcgtattat gtacgtccca gaggaagatc ttttcgagtt tgctctcgac 3900

tacaaaaact tttcgcggac cgatgcggat tacattaaaa aatggaaact ctattcgtac 3960

ggcaacagaa tcaggatttt tcgcaaccct aagaagaata acgtctttga ttgggaggaa 4020

gtttgcttga ctagcgcgta caaggagctc tttaataagt atggcattaa ctaccaacag 4080

ggtgatatca gagcactgct ttgcgaacaa tctgacaagg ctttctactc atccttcatg 4140

gctttgatga gcctgatgct ccagatgaga aattcaatta caggcagaac cgacgtggat 4200

ttcttgatct ccccggttaa aaattctgat ggcatctttt acgatagcag gaactatgaa 4260

gcgcaagaga atgcgattct gccaaaaaat gcagacgcca acggtgccta taacatcgcc 4320

aggaaagtcc tgtgggcgat cggccagttc aaaaaggccg aagacgaaaa attggacaag 4380

gtcaaaatcg ctatcagcaa caaagagtgg ctggagtatg ctcagacatc cgtaaagcat 4440

acgcgtgact ccggcggcag caccaacctg tccgacatca tcgagaagga gacgggcaag 4500

caactcgtga tccaggagag catcctcatg ctgccagagg aggtggagga ggtcatcggc 4560

aacaagccag agtccgacat cctggtgcac accgcctacg acgagtccac cgacgagaac 4620

gtcatgctcc tgaccagcga cgccccagag tacaagccat gggccctcgt catccaggac 4680

agcaacgggg agaacaagat caagatgctg tcggggggga gcccaaagaa gaagcggaag 4740

gtgagcggcg gcagcccaaa gaagaagcgc aaggtctag 4779

<210> 10

<211> 6305

<212> DNA

<213> Artificial Sequence

<220>

<223> BDUBI10-dLBCPF1-PBE-2NLS

<400> 10

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

tcgacgagtc taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag 660

cagacggcac ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg 720

ttggacttgc tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg 780

gcacggcagg cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc 840

caccgctcct tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc 900

ctctttcccc aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa 960

tccacccgtc ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc 1020

taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc 1080

atgtttgtgt tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg 1140

cgacctgtac gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc 1200

ctgggatggc tctagccgtt ccgcagacgg gatcgatcta ggataggtat acatgttgat 1260

gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 1320

cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 1380

acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 1440

ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 1500

tctgcaggtc gaagcttgaa gcaaacatgc caaagaagaa gaggaaggtt tcatcggaga 1560

ccggccctgt tgctgttgac cccaccctgc ggcggagaat cgagccacac gagttcgagg 1620

tgttcttcga cccaagggag ctccgcaagg agacgtgcct cctgtacgag atcaactggg 1680

gcggcaggca ctccatctgg aggcacacca gccaaaacac caacaagcac gtggaggtca 1740

acttcatcga gaagttcacc accgagaggt acttctgccc aaacacccgc tgctccatca 1800

cctggttcct gtcctggagc ccatgcggcg agtgctccag ggccatcacc gagttcctca 1860

gccgctaccc acacgtcacc ctgttcatct acatcgccag gctctaccac cacgccgacc 1920

caaggaacag gcagggcctc cgcgacctga tctccagcgg cgtgaccatc caaatcatga 1980

ccgagcagga gtccggctac tgctggagga acttcgtcaa ctactcccca agcaacgagg 2040

cccactggcc aaggtaccca cacctctggg tgcgcctcta cgtgctcgag ctgtactgca 2100

tcatcctcgg cctgccacca tgcctcaaca tcctgaggcg caagcaacca cagctgacct 2160

tcttcaccat cgccctccaa agctgccact accagaggct cccaccacac atcctgtggg 2220

ctaccggcct caagtccggc agcgagacgc caggcacctc cgagagcgct acgcctgaac 2280

ttaagtcaaa gctcgagaaa ttcaccaact gttattcgtt gagcaaaaca ctgcggttta 2340

aagcgattcc agtcggcaag actcaagaga atatagacaa taagcggctg ttggtggaag 2400

atgaaaagcg cgcggaagac tacaaagggg tgaagaagtt gttggacaga tactacctct 2460

cttttatcaa tgatgtcttg cactcaatca aattgaagaa tctgaacaac tacatctccc 2520

tcttcagaaa gaaaacaagg acagaaaagg agaataagga acttgaaaat ttggagatca 2580

atctgaggaa agagatcgcg aaagccttta aaggcaacga aggatacaaa agtctgttca 2640

agaaggatat aattgagaca attttgccag agttcctcga tgacaaggac gagattgcgc 2700

tggtcaattc gttcaacgga ttcacaacag cattcacagg cttctttgat aatcgggaaa 2760

atatgttctc tgaggaggca aagtccactt ctattgcgtt caggtgtatc aatgagaatc 2820

tcactaggta catttccaac atggatatct ttgagaaggt tgacgcaatt tttgacaagc 2880

acgaagttca ggagattaag gagaagatcc tcaattccga ttatgacgtt gaggacttct 2940

tcgaaggtga gttttttaat ttcgtgctca ctcaagaggg tatcgacgtg tataatgcga 3000

tcatcggtgg gttcgtgact gagtccggtg aaaagattaa gggattgaac gagtatatca 3060

acctttacaa ccaaaagacg aaacagaagc tgccaaagtt caagcctctt tacaaacagg 3120

ttctttcaga ccgcgagtca ctctcgttct atggggaggg ctacacttcg gatgaggaag 3180

tcctggaggt gttcaggaat actctcaata agaattcgga gattttctct tctataaaaa 3240

aactggaaaa gttgtttaag aattttgacg aatactctag cgccggcata tttgtgaaaa 3300

acggcccggc catatcaacg ataagtaaag atatcttcgg cgaatggaac gtgatcagag 3360

acaaatggaa cgcggagtat gacgatattc acctgaagaa gaaggctgtc gtaacggaga 3420

agtacgagga tgatcgcagg aaaagcttca aaaagatcgg aagtttcagc ctggaacagt 3480

tgcaggagta tgctgacgcc gatcttagcg tcgtcgagaa gttgaaggag ataatcatcc 3540

aaaaggtcga cgagatatat aaagtctatg gatcaagtga aaaactgttc gacgccgact 3600

tcgttttgga gaagtccctg aagaagaacg acgctgttgt tgccattatg aaggatctgc 3660

tcgacagcgt gaagagtttc gagaactata ttaaggcttt tttcggggag gggaaggaga 3720

ctaacagaga tgagtccttc tacggagact tcgtcctcgc gtacgatata ctccttaagg 3780

tagaccacat ctacgacgca atcagaaatt acgtgacaca aaagccgtac agcaaggaca 3840

agttcaaact ctacttccag aacccccagt tcatgggcgg ctgggacaag gacaaggaaa 3900

cggattacag ggctacgatc ctgaggtatg gttcaaaata ctacttggcg attatggaca 3960

agaagtacgc caagtgtctc cagaagattg acaaagacga tgtcaatggc aattatgaga 4020

agatcaacta caagctgctt ccgggtccga acaagatgct cccaaaggtt ttcttcagca 4080

agaaatggat ggcctactat aacccaagcg aggacatcca gaagatttat aagaacggta 4140

cgttcaagaa gggcgacatg ttcaatctta acgactgtca caagctgatc gacttcttca 4200

aagactcaat tagccggtac ccaaagtggt ctaacgccta tgacttcaac ttttcggaaa 4260

ccgagaagta caaggatata gccggatttt atagagaggt ggaagagcag ggctacaagg 4320

tgtcattcga gtccgccagc aagaaggaag tggacaagct cgtggaagag ggtaagctct 4380

acatgttcca gatttataat aaagacttta gcgataagag ccacgggaca cctaatctcc 4440

acacaatgta tttcaagctg ctcttcgacg agaataacca cggccaaatc aggttgtcag 4500

gaggggctga actcttcatg cggcgcgcta gccttaagaa ggaggagctt gtagtccacc 4560

ctgcgaatag tccaattgcg aataagaacc cggacaatcc taaaaagact acaacattga 4620

gctacgacgt gtacaaggat aagaggtttt ccgaggatca gtacgagctc cacatcccga 4680

ttgcgatcaa caagtgccca aagaatattt tcaagataaa cacagaggtg cgtgtactcc 4740

tgaagcatga cgacaatcct tacgtcattg ggattgctcg gggcgagagg aacctcctct 4800

atattgtggt ggtggacggg aaggggaaca tagtcgaaca gtactccctt aacgaaataa 4860

ttaacaattt caacggcatc cgtatcaaga ccgactacca ttcgttgctg gacaagaagg 4920

agaaggagag atttgaggcg cggcaaaatt ggacaagtat cgagaacatc aaggaactca 4980

aagcaggtta tatctctcaa gttgtgcata agatatgcga gctggttgag aagtatgacg 5040

cagtgatcgc tcttgaggac ctcaactcgg gctttaagaa ttctagagtt aaagtggaga 5100

agcaggtcta tcaaaagttc gagaagatgc ttatagataa gctcaactac atggtcgata 5160

agaaatcgaa cccatgtgcc accggcggcg cactcaaagg ttaccaaata acaaacaaat 5220

tcgagtcctt caaatcgatg agtactcaga atgggttcat attttatata ccggcgtggc 5280

ttacgtctaa gatcgacccg tcaactggtt ttgtcaacct gttgaagacg aaatacacgt 5340

ccattgccga ttcgaaaaag ttcatatcta gttttgatcg tattatgtac gtcccagagg 5400

aagatctttt cgagtttgct ctcgactaca aaaacttttc gcggaccgat gcggattaca 5460

ttaaaaaatg gaaactctat tcgtacggca acagaatcag gatttttcgc aaccctaaga 5520

agaataacgt ctttgattgg gaggaagttt gcttgactag cgcgtacaag gagctcttta 5580

ataagtatgg cattaactac caacagggtg atatcagagc actgctttgc gaacaatctg 5640

acaaggcttt ctactcatcc ttcatggctt tgatgagcct gatgctccag atgagaaatt 5700

caattacagg cagaaccgac gtggatttct tgatctcccc ggttaaaaat tctgatggca 5760

tcttttacga tagcaggaac tatgaagcgc aagagaatgc gattctgcca aaaaatgcag 5820

acgccaacgg tgcctataac atcgccagga aagtcctgtg ggcgatcggc cagttcaaaa 5880

aggccgaaga cgaaaaattg gacaaggtca aaatcgctat cagcaacaaa gagtggctgg 5940

agtatgctca gacatccgta aagcatacgc gtgactccgg cggcagcacc aacctgtccg 6000

acatcatcga gaaggagacg ggcaagcaac tcgtgatcca ggagagcatc ctcatgctgc 6060

cagaggaggt ggaggaggtc atcggcaaca agccagagtc cgacatcctg gtgcacaccg 6120

cctacgacga gtccaccgac gagaacgtca tgctcctgac cagcgacgcc ccagagtaca 6180

agccatgggc cctcgtcatc caggacagca acggggagaa caagatcaag atgctgtcgg 6240

gggggagccc aaagaagaag cggaaggtga gcggcggcag cccaaagaag aagcgcaagg 6300

tctag 6305

<210> 11

<211> 5196

<212> DNA

<213> Artificial Sequence

<220>

<223> dFNCPF1-ABE7.10-2NLS

<400> 11

atgccaaaaa agaagagaaa ggtttcaggc ggctcctccg aggtggagtt ctctcacgag 60

tattggatga ggcacgctct tacacttgct aagagagctt gggacgaaag agaagtgcca 120

gttggcgccg ttcttgtgca taataatagg gtgatcggcg agggttggaa tagaccaatt 180

ggaaggcatg atccaacagc tcacgcagag attatggctc tcagacaagg cggcctcgtt 240

atgcagaact acaggctcat tgacgctaca ctctacgtga cactcgaacc ttgcgttatg 300

tgcgccggag ctatgattca ttctaggatt ggcagggtcg tgtttggagc tagggacgct 360

aaaacaggag ccgccggatc tcttatggac gtgttgcatc atccaggcat gaaccatagg 420

gtggagatta cagagggcat tcttgcagac gagtgcgctg ctcttctttc cgatttcttc 480

aggatgagaa ggcaggagat taaggcccag aagaaggctc agtcttctac agatagcgga 540

ggatcttccg gaggatctag cggctccgag acaccaggaa catccgaaag cgctacacca 600

gaatctagcg gaggctcttc cggaggatct tctgaagtgg agttctccca cgagtattgg 660

atgaggcacg ctcttacact tgctaaaagg gctagggacg aaagggaagt tccagttgga 720

gctgttctcg tgctcaataa cagggtgatt ggcgagggtt ggaatagagc cattggactc 780

catgatccaa cagctcacgc agagattatg gctcttagac aaggcggcct cgttatgcag 840

aattacagac tcatcgacgc cacactctac gttaccttcg aaccttgcgt tatgtgcgcc 900

ggagctatga tccattctag gattggcagg gtcgtgttcg gcgttagaaa cgctaagaca 960

ggagctgcag gctctcttat ggacgttctt cattacccag gcatgaatca tagagtggag 1020

atcacagaag gcattcttgc agacgagtgc gcagctctcc tttgctattt cttcaggatg 1080

ccgaggcaag ttttcaacgc tcagaagaag gcccagtctt ctacagattc cggcggatct 1140

tctggaggat ctagcggctc cgagacacca ggaacatccg aatccgctac accagagtct 1200

tctggaggat ctagcggagg atctcttaag tccatctacc aggagttcgt caataagtac 1260

tcactctcta agaccctgcg gttcgagctg atcccgcagg gcaagacact cgagaacatc 1320

aaggcgcgcg gcctgattct cgacgatgag aagcgggcca aggactacaa gaaggcgaag 1380

cagatcattg ataagtacca ccagttcttc atcgaggaga ttctgtccag cgtgtgcatc 1440

tctgaggatc tcctgcagaa ttactccgac gtctacttca agctcaagaa gtctgacgat 1500

gacaacctgc agaaggattt caagtccgcc aaggacacca tcaagaagca gatttctgag 1560

tacatcaagg attccgagaa gttcaagaat ctcttcaacc agaatctgat tgatgcgaag 1620

aagggccagg agtctgacct gatcctctgg ctgaagcagt ccaaggacaa tggcattgag 1680

ctgttcaagg ccaacagcga tatcaccgat attgacgagg cgctggagat cattaagtca 1740

ttcaagggct ggaccacata cttcaagggc ttccatgaga accggaagaa tgtgtactca 1800

tctaacgaca ttccgacctc catcatctac aggatcgtcg atgacaatct gccaaagttc 1860

ctcgagaaca aggccaagta cgagtccctc aaggacaagg ccccggaggc gattaattac 1920

gagcagatca agaaggatct ggcggaggag ctgaccttcg atatcgacta caagacaagc 1980

gaggtgaacc agagggtgtt ctccctcgat gaggtgttcg agatcgccaa tttcaacaat 2040

tacctgaacc agtccggcat taccaagttc aatacaatca ttggcggcaa gttcgtcaac 2100

ggcgagaata ccaagcgcaa gggcattaac gagtacatca atctctactc ccagcagatc 2160

aacgacaaga ccctgaagaa gtacaagatg tctgtgctct tcaagcagat cctgtccgat 2220

acagagtcca agagcttcgt cattgataag ctcgaggacg acagcgacgt ggtcaccaca 2280

atgcagtcat tctacgagca gatcgccgcg ttcaagaccg tggaggagaa gagcattaag 2340

gagacactct cactcctgtt cgatgacctg aaggcccaga agctcgacct gagcaagatc 2400

tacttcaaga acgataagag cctcacagac ctgtcacagc aggtgttcga tgactactca 2460

gtgattggca ccgccgtcct cgagtacatt acacagcaga tcgcgccaaa gaacctcgat 2520

aatccttcta agaaggagca ggagctgatc gccaagaaaa ccgagaaggc gaagtacctc 2580

tccctggaga caattaagct cgccctggag gagttcaata agcacaggga tattgacaag 2640

cagtgccgct tcgaggagat cctcgcgaac ttcgccgcga tcccaatgat tttcgatgag 2700

atcgcccaga acaaggacaa tctggcgcag atctctatta agtaccagaa ccagggcaag 2760

aaggacctcc tgcaggcctc cgcagaggac gacgtgaagg ccatcaagga tctcctggac 2820

cagaccaaca atctcctgca caagctcaag atcttccata tttcacagtc tgaggataag 2880

gccaatatcc tcgataagga cgagcatttc tacctggtgt tcgaggagtg ctacttcgag 2940

ctggcgaaca ttgtccctct gtacaacaag attaggaatt acatcacaca gaagccgtac 3000

agcgacgaga agttcaagct caacttcgag aattcaaccc tggccaacgg ctgggataag 3060

aataaggagc ctgacaacac agcgatcctc ttcatcaagg acgacaagta ctacctgggc 3120

gtgatgaata agaagaacaa taagatcttc gatgacaagg ccattaagga gaacaagggc 3180

gagggctaca agaagatcgt gtacaagctc ctgcctggcg ccaataagat gctcccgaag 3240

gtgttcttct ccgcgaagtc cattaagttc tacaacccaa gcgaggatat cctcaggatc 3300

aggaaccact ctacccatac aaagaacggc tcccctcaga agggctacga gaagttcgag 3360

ttcaatatcg aggattgccg gaagttcatt gacttctaca agcagtccat cagcaagcac 3420

cctgagtgga aggatttcgg cttccgcttc agcgacaccc agcggtacaa ctcaatcgat 3480

gagttctaca gggaggtgga gaatcagggc tacaagctca cattcgagaa catttcagag 3540

tcttacatcg actccgtggt caatcagggc aagctctacc tgttccagat ctacaacaag 3600

gatttcagcg cctactcaaa gggcaggccg aacctccata ccctgtactg gaaggcgctc 3660

ttcgatgagc gcaatctgca ggacgtggtc tacaagctca acggcgaggc cgagctgttc 3720

taccgcaagc agtctattcc gaagaagatc acacacccag cgaaggaggc catcgcgaac 3780

aagaataagg acaatccgaa gaaggagtcc gtgttcgagt acgatctcat taaggacaag 3840

cggttcaccg aggataagtt cttcttccat tgcccaatca caattaactt caagtccagc 3900

ggcgccaaca agttcaatga cgagatcaat ctcctgctca aggagaaggc gaacgatgtg 3960

catatcctct ccattgcccg cggcgagcgg catctcgcct actacaccct ggtcgatggc 4020

aagggcaata tcattaagca ggataccttc aacatcattg gcaatgaccg catgaaaacc 4080

aactaccacg ataagctcgc cgcgatcgag aaggataggg acagcgcgcg caaggactgg 4140

aagaagatca acaatattaa ggagatgaag gagggctacc tctcacaggt ggtccatgag 4200

attgccaagc tcgtgatcga gtacaatgcg attgtggtgt tcgaggacct caacttcggc 4260

ttcaagcggg gcaggttcaa ggtggagaag caggtctacc agaagctgga gaagatgctc 4320

atcgagaagc tcaattacct ggtgttcaag gataacgagt tcgacaagac cggaggagtc 4380

ctcagggcat accagctgac cgcgccattc gagacattca agaagatggg caagcagaca 4440

ggcatcatct actacgtgcc agccggcttc acctccaaga tctgccctgt gacaggcttc 4500

gtcaaccagc tctaccctaa gtacgagtcc gtcagcaagt cacaggagtt cttcagcaag 4560

ttcgataaga tttgctacaa tctggacaag ggctacttcg agttctcatt cgattacaag 4620

aacttcggcg acaaggccgc gaagggcaag tggaccattg cgtctttcgg ctcccggctc 4680

atcaacttca ggaatagcga taagaaccac aattgggaca ccagggaggt gtacccgaca 4740

aaggagctgg agaagctgct caaggactac tcaatcgagt acggccatgg cgagtgcatt 4800

aaggccgcga tctgcggcga gagcgataag aagttcttcg ccaagctgac ctcagtcctc 4860

aatacaatcc tgcagatgag gaactctaag accggcacag agctggacta cctgatttcc 4920

ccagtggccg atgtcaacgg caatttcttc gacagccgcc aggcgccgaa gaatatgcca 4980

caggacgccg acgccaacgg cgcataccac atcggcctca agggcctgat gctgctcgga 5040

cgcattaaga acaatcagga gggcaagaag ctcaacctcg tgatcaagaa cgaggagtac 5100

ttcgagttcg tccaaaaccg gaacaattcc ggcggcagcc caaagaagaa gaggaaggtg 5160

agcggcggca gcccaaagaa gaagcgcaag gtctag 5196

<210> 12

<211> 4980

<212> DNA

<213> Artificial Sequence

<220>

<223> dLBCPF1-ABE7.10-2NLS

<400> 12

atgccaaaaa agaagagaaa ggtttcaggc ggctcctccg aggtggagtt ctctcacgag 60

tattggatga ggcacgctct tacacttgct aagagagctt gggacgaaag agaagtgcca 120

gttggcgccg ttcttgtgca taataatagg gtgatcggcg agggttggaa tagaccaatt 180

ggaaggcatg atccaacagc tcacgcagag attatggctc tcagacaagg cggcctcgtt 240

atgcagaact acaggctcat tgacgctaca ctctacgtga cactcgaacc ttgcgttatg 300

tgcgccggag ctatgattca ttctaggatt ggcagggtcg tgtttggagc tagggacgct 360

aaaacaggag ccgccggatc tcttatggac gtgttgcatc atccaggcat gaaccatagg 420

gtggagatta cagagggcat tcttgcagac gagtgcgctg ctcttctttc cgatttcttc 480

aggatgagaa ggcaggagat taaggcccag aagaaggctc agtcttctac agatagcgga 540

ggatcttccg gaggatctag cggctccgag acaccaggaa catccgaaag cgctacacca 600

gaatctagcg gaggctcttc cggaggatct tctgaagtgg agttctccca cgagtattgg 660

atgaggcacg ctcttacact tgctaaaagg gctagggacg aaagggaagt tccagttgga 720

gctgttctcg tgctcaataa cagggtgatt ggcgagggtt ggaatagagc cattggactc 780

catgatccaa cagctcacgc agagattatg gctcttagac aaggcggcct cgttatgcag 840

aattacagac tcatcgacgc cacactctac gttaccttcg aaccttgcgt tatgtgcgcc 900

ggagctatga tccattctag gattggcagg gtcgtgttcg gcgttagaaa cgctaagaca 960

ggagctgcag gctctcttat ggacgttctt cattacccag gcatgaatca tagagtggag 1020

atcacagaag gcattcttgc agacgagtgc gcagctctcc tttgctattt cttcaggatg 1080

ccgaggcaag ttttcaacgc tcagaagaag gcccagtctt ctacagattc cggcggatct 1140

tctggaggat ctagcggctc cgagacacca ggaacatccg aatccgctac accagagtct 1200

tctggaggat ctagcggagg atctcttaag tcaaagctcg agaaattcac caactgttat 1260

tcgttgagca aaacactgcg gtttaaagcg attccagtcg gcaagactca agagaatata 1320

gacaataagc ggctgttggt ggaagatgaa aagcgcgcgg aagactacaa aggggtgaag 1380

aagttgttgg acagatacta cctctctttt atcaatgatg tcttgcactc aatcaaattg 1440

aagaatctga acaactacat ctccctcttc agaaagaaaa caaggacaga aaaggagaat 1500

aaggaacttg aaaatttgga gatcaatctg aggaaagaga tcgcgaaagc ctttaaaggc 1560

aacgaaggat acaaaagtct gttcaagaag gatataattg agacaatttt gccagagttc 1620

ctcgatgaca aggacgagat tgcgctggtc aattcgttca acggattcac aacagcattc 1680

acaggcttct ttgataatcg ggaaaatatg ttctctgagg aggcaaagtc cacttctatt 1740

gcgttcaggt gtatcaatga gaatctcact aggtacattt ccaacatgga tatctttgag 1800

aaggttgacg caatttttga caagcacgaa gttcaggaga ttaaggagaa gatcctcaat 1860

tccgattatg acgttgagga cttcttcgaa ggtgagtttt ttaatttcgt gctcactcaa 1920

gagggtatcg acgtgtataa tgcgatcatc ggtgggttcg tgactgagtc cggtgaaaag 1980

attaagggat tgaacgagta tatcaacctt tacaaccaaa agacgaaaca gaagctgcca 2040

aagttcaagc ctctttacaa acaggttctt tcagaccgcg agtcactctc gttctatggg 2100

gagggctaca cttcggatga ggaagtcctg gaggtgttca ggaatactct caataagaat 2160

tcggagattt tctcttctat aaaaaaactg gaaaagttgt ttaagaattt tgacgaatac 2220

tctagcgccg gcatatttgt gaaaaacggc ccggccatat caacgataag taaagatatc 2280

ttcggcgaat ggaacgtgat cagagacaaa tggaacgcgg agtatgacga tattcacctg 2340

aagaagaagg ctgtcgtaac ggagaagtac gaggatgatc gcaggaaaag cttcaaaaag 2400

atcggaagtt tcagcctgga acagttgcag gagtatgctg acgccgatct tagcgtcgtc 2460

gagaagttga aggagataat catccaaaag gtcgacgaga tatataaagt ctatggatca 2520

agtgaaaaac tgttcgacgc cgacttcgtt ttggagaagt ccctgaagaa gaacgacgct 2580

gttgttgcca ttatgaagga tctgctcgac agcgtgaaga gtttcgagaa ctatattaag 2640

gcttttttcg gggaggggaa ggagactaac agagatgagt ccttctacgg agacttcgtc 2700

ctcgcgtacg atatactcct taaggtagac cacatctacg acgcaatcag aaattacgtg 2760

acacaaaagc cgtacagcaa ggacaagttc aaactctact tccagaaccc ccagttcatg 2820

ggcggctggg acaaggacaa ggaaacggat tacagggcta cgatcctgag gtatggttca 2880

aaatactact tggcgattat ggacaagaag tacgccaagt gtctccagaa gattgacaaa 2940

gacgatgtca atggcaatta tgagaagatc aactacaagc tgcttccggg tccgaacaag 3000

atgctcccaa aggttttctt cagcaagaaa tggatggcct actataaccc aagcgaggac 3060

atccagaaga tttataagaa cggtacgttc aagaagggcg acatgttcaa tcttaacgac 3120

tgtcacaagc tgatcgactt cttcaaagac tcaattagcc ggtacccaaa gtggtctaac 3180

gcctatgact tcaacttttc ggaaaccgag aagtacaagg atatagccgg attttataga 3240

gaggtggaag agcagggcta caaggtgtca ttcgagtccg ccagcaagaa ggaagtggac 3300

aagctcgtgg aagagggtaa gctctacatg ttccagattt ataataaaga ctttagcgat 3360

aagagccacg ggacacctaa tctccacaca atgtatttca agctgctctt cgacgagaat 3420

aaccacggcc aaatcaggtt gtcaggaggg gctgaactct tcatgcggcg cgctagcctt 3480

aagaaggagg agcttgtagt ccaccctgcg aatagtccaa ttgcgaataa gaacccggac 3540

aatcctaaaa agactacaac attgagctac gacgtgtaca aggataagag gttttccgag 3600

gatcagtacg agctccacat cccgattgcg atcaacaagt gcccaaagaa tattttcaag 3660

ataaacacag aggtgcgtgt actcctgaag catgacgaca atccttacgt cattgggatt 3720

gctcggggcg agaggaacct cctctatatt gtggtggtgg acgggaaggg gaacatagtc 3780

gaacagtact cccttaacga aataattaac aatttcaacg gcatccgtat caagaccgac 3840

taccattcgt tgctggacaa gaaggagaag gagagatttg aggcgcggca aaattggaca 3900

agtatcgaga acatcaagga actcaaagca ggttatatct ctcaagttgt gcataagata 3960

tgcgagctgg ttgagaagta tgacgcagtg atcgctcttg aggacctcaa ctcgggcttt 4020

aagaattcta gagttaaagt ggagaagcag gtctatcaaa agttcgagaa gatgcttata 4080

gataagctca actacatggt cgataagaaa tcgaacccat gtgccaccgg cggcgcactc 4140

aaaggttacc aaataacaaa caaattcgag tccttcaaat cgatgagtac tcagaatggg 4200

ttcatatttt atataccggc gtggcttacg tctaagatcg acccgtcaac tggttttgtc 4260

aacctgttga agacgaaata cacgtccatt gccgattcga aaaagttcat atctagtttt 4320

gatcgtatta tgtacgtccc agaggaagat cttttcgagt ttgctctcga ctacaaaaac 4380

ttttcgcgga ccgatgcgga ttacattaaa aaatggaaac tctattcgta cggcaacaga 4440

atcaggattt ttcgcaaccc taagaagaat aacgtctttg attgggagga agtttgcttg 4500

actagcgcgt acaaggagct ctttaataag tatggcatta actaccaaca gggtgatatc 4560

agagcactgc tttgcgaaca atctgacaag gctttctact catccttcat ggctttgatg 4620

agcctgatgc tccagatgag aaattcaatt acaggcagaa ccgacgtgga tttcttgatc 4680

tccccggtta aaaattctga tggcatcttt tacgatagca ggaactatga agcgcaagag 4740

aatgcgattc tgccaaaaaa tgcagacgcc aacggtgcct ataacatcgc caggaaagtc 4800

ctgtgggcga tcggccagtt caaaaaggcc gaagacgaaa aattggacaa ggtcaaaatc 4860

gctatcagca acaaagagtg gctggagtat gctcagacat ccgtaaagca ttccggcggc 4920

agcccaaaga agaagaggaa ggtgagcggc ggcagcccaa agaagaagcg caaggtctag 4980

<210> 13

<211> 6506

<212> DNA

<213> Artificial Sequence

<220>

<223> dLBCPF1-ABE2-X

<400> 13

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

tcgacgagtc taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag 660

cagacggcac ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg 720

ttggacttgc tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg 780

gcacggcagg cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc 840

caccgctcct tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc 900

ctctttcccc aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa 960

tccacccgtc ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc 1020

taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc 1080

atgtttgtgt tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg 1140

cgacctgtac gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc 1200

ctgggatggc tctagccgtt ccgcagacgg gatcgatcta ggataggtat acatgttgat 1260

gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 1320

cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 1380

acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 1440

ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 1500

tctgcaggtc gaagcttgaa gcaaacatgc caaaaaagaa gagaaaggtt tcaggcggct 1560

cctccgaggt ggagttctct cacgagtatt ggatgaggca cgctcttaca cttgctaaga 1620

gagcttggga cgaaagagaa gtgccagttg gcgccgttct tgtgcataat aatagggtga 1680

tcggcgaggg ttggaataga ccaattggaa ggcatgatcc aacagctcac gcagagatta 1740

tggctctcag acaaggcggc ctcgttatgc agaactacag gctcattgac gctacactct 1800

acgtgacact cgaaccttgc gttatgtgcg ccggagctat gattcattct aggattggca 1860

gggtcgtgtt tggagctagg gacgctaaaa caggagccgc cggatctctt atggacgtgt 1920

tgcatcatcc aggcatgaac catagggtgg agattacaga gggcattctt gcagacgagt 1980

gcgctgctct tctttccgat ttcttcagga tgagaaggca ggagattaag gcccagaaga 2040

aggctcagtc ttctacagat agcggaggat cttccggagg atctagcggc tccgagacac 2100

caggaacatc cgaaagcgct acaccagaat ctagcggagg ctcttccgga ggatcttctg 2160

aagtggagtt ctcccacgag tattggatga ggcacgctct tacacttgct aaaagggcta 2220

gggacgaaag ggaagttcca gttggagctg ttctcgtgct caataacagg gtgattggcg 2280

agggttggaa tagagccatt ggactccatg atccaacagc tcacgcagag attatggctc 2340

ttagacaagg cggcctcgtt atgcagaatt acagactcat cgacgccaca ctctacgtta 2400

ccttcgaacc ttgcgttatg tgcgccggag ctatgatcca ttctaggatt ggcagggtcg 2460

tgttcggcgt tagaaacgct aagacaggag ctgcaggctc tcttatggac gttcttcatt 2520

acccaggcat gaatcataga gtggagatca cagaaggcat tcttgcagac gagtgcgcag 2580

ctctcctttg ctatttcttc aggatgccga ggcaagtttt caacgctcag aagaaggccc 2640

agtcttctac agattccggc ggatcttctg gaggatctag cggctccgag acaccaggaa 2700

catccgaatc cgctacacca gagtcttctg gaggatctag cggaggatct cttaagtcaa 2760

agctcgagaa attcaccaac tgttattcgt tgagcaaaac actgcggttt aaagcgattc 2820

cagtcggcaa gactcaagag aatatagaca ataagcggct gttggtggaa gatgaaaagc 2880

gcgcggaaga ctacaaaggg gtgaagaagt tgttggacag atactacctc tcttttatca 2940

atgatgtctt gcactcaatc aaattgaaga atctgaacaa ctacatctcc ctcttcagaa 3000

agaaaacaag gacagaaaag gagaataagg aacttgaaaa tttggagatc aatctgagga 3060

aagagatcgc gaaagccttt aaaggcaacg aaggatacaa aagtctgttc aagaaggata 3120

taattgagac aattttgcca gagttcctcg atgacaagga cgagattgcg ctggtcaatt 3180

cgttcaacgg attcacaaca gcattcacag gcttctttga taatcgggaa aatatgttct 3240

ctgaggaggc aaagtccact tctattgcgt tcaggtgtat caatgagaat ctcactaggt 3300

acatttccaa catggatatc tttgagaagg ttgacgcaat ttttgacaag cacgaagttc 3360

aggagattaa ggagaagatc ctcaattccg attatgacgt tgaggacttc ttcgaaggtg 3420

agttttttaa tttcgtgctc actcaagagg gtatcgacgt gtataatgcg atcatcggtg 3480

ggttcgtgac tgagtccggt gaaaagatta agggattgaa cgagtatatc aacctttaca 3540

accaaaagac gaaacagaag ctgccaaagt tcaagcctct ttacaaacag gttctttcag 3600

accgcgagtc actctcgttc tatggggagg gctacacttc ggatgaggaa gtcctggagg 3660

tgttcaggaa tactctcaat aagaattcgg agattttctc ttctataaaa aaactggaaa 3720

agttgtttaa gaattttgac gaatactcta gcgccggcat atttgtgaaa aacggcccgg 3780

ccatatcaac gataagtaaa gatatcttcg gcgaatggaa cgtgatcaga gacaaatgga 3840

acgcggagta tgacgatatt cacctgaaga agaaggctgt cgtaacggag aagtacgagg 3900

atgatcgcag gaaaagcttc aaaaagatcg gaagtttcag cctggaacag ttgcaggagt 3960

atgctgacgc cgatcttagc gtcgtcgaga agttgaagga gataatcatc caaaaggtcg 4020

acgagatata taaagtctat ggatcaagtg aaaaactgtt cgacgccgac ttcgttttgg 4080

agaagtccct gaagaagaac gacgctgttg ttgccattat gaaggatctg ctcgacagcg 4140

tgaagagttt cgagaactat attaaggctt ttttcgggga ggggaaggag actaacagag 4200

atgagtcctt ctacggagac ttcgtcctcg cgtacgatat actccttaag gtagaccaca 4260

tctacgacgc aatcagaaat tacgtgacac aaaagccgta cagcaaggac aagttcaaac 4320

tctacttcca gaacccccag ttcatgggcg gctgggacaa ggacaaggaa acggattaca 4380

gggctacgat cctgaggtat ggttcaaaat actacttggc gattatggac aagaagtacg 4440

ccaagtgtct ccagaagatt gacaaagacg atgtcaatgg caattatgag aagatcaact 4500

acaagctgct tccgggtccg aacaagatgc tcccaaaggt tttcttcagc aagaaatgga 4560

tggcctacta taacccaagc gaggacatcc agaagattta taagaacggt acgttcaaga 4620

agggcgacat gttcaatctt aacgactgtc acaagctgat cgacttcttc aaagactcaa 4680

ttagccggta cccaaagtgg tctaacgcct atgacttcaa cttttcggaa accgagaagt 4740

acaaggatat agccggattt tatagagagg tggaagagca gggctacaag gtgtcattcg 4800

agtccgccag caagaaggaa gtggacaagc tcgtggaaga gggtaagctc tacatgttcc 4860

agatttataa taaagacttt agcgataaga gccacgggac acctaatctc cacacaatgt 4920

atttcaagct gctcttcgac gagaataacc acggccaaat caggttgtca ggaggggctg 4980

aactcttcat gcggcgcgct agccttaaga aggaggagct tgtagtccac cctgcgaata 5040

gtccaattgc gaataagaac ccggacaatc ctaaaaagac tacaacattg agctacgacg 5100

tgtacaagga taagaggttt tccgaggatc agtacgagct ccacatcccg attgcgatca 5160

acaagtgccc aaagaatatt ttcaagataa acacagaggt gcgtgtactc ctgaagcatg 5220

acgacaatcc ttacgtcatt gggattgctc ggggcgagag gaacctcctc tatattgtgg 5280

tggtggacgg gaaggggaac atagtcgaac agtactccct taacgaaata attaacaatt 5340

tcaacggcat ccgtatcaag accgactacc attcgttgct ggacaagaag gagaaggaga 5400

gatttgaggc gcggcaaaat tggacaagta tcgagaacat caaggaactc aaagcaggtt 5460

atatctctca agttgtgcat aagatatgcg agctggttga gaagtatgac gcagtgatcg 5520

ctcttgagga cctcaactcg ggctttaaga attctagagt taaagtggag aagcaggtct 5580

atcaaaagtt cgagaagatg cttatagata agctcaacta catggtcgat aagaaatcga 5640

acccatgtgc caccggcggc gcactcaaag gttaccaaat aacaaacaaa ttcgagtcct 5700

tcaaatcgat gagtactcag aatgggttca tattttatat accggcgtgg cttacgtcta 5760

agatcgaccc gtcaactggt tttgtcaacc tgttgaagac gaaatacacg tccattgccg 5820

attcgaaaaa gttcatatct agttttgatc gtattatgta cgtcccagag gaagatcttt 5880

tcgagtttgc tctcgactac aaaaactttt cgcggaccga tgcggattac attaaaaaat 5940

ggaaactcta ttcgtacggc aacagaatca ggatttttcg caaccctaag aagaataacg 6000

tctttgattg ggaggaagtt tgcttgacta gcgcgtacaa ggagctcttt aataagtatg 6060

gcattaacta ccaacagggt gatatcagag cactgctttg cgaacaatct gacaaggctt 6120

tctactcatc cttcatggct ttgatgagcc tgatgctcca gatgagaaat tcaattacag 6180

gcagaaccga cgtggatttc ttgatctccc cggttaaaaa ttctgatggc atcttttacg 6240

atagcaggaa ctatgaagcg caagagaatg cgattctgcc aaaaaatgca gacgccaacg 6300

gtgcctataa catcgccagg aaagtcctgt gggcgatcgg ccagttcaaa aaggccgaag 6360

acgaaaaatt ggacaaggtc aaaatcgcta tcagcaacaa agagtggctg gagtatgctc 6420

agacatccgt aaagcattcc ggcggcagcc caaagaagaa gaggaaggtg agcggcggca 6480

gcccaaagaa gaagcgcaag gtctag 6506

<210> 14

<211> 4971

<212> DNA

<213> Artificial Sequence

<220>

<223> LBCPF1-ABE2-X2

<400> 14

atgtcaaagc tcgagaaatt caccaactgt tattcgttga gcaaaacact gcggtttaaa 60

gcgattccag tcggcaagac tcaagagaat atagacaata agcggctgtt ggtggaagat 120

gaaaagcgcg cggaagacta caaaggggtg aagaagttgt tggacagata ctacctctct 180

tttatcaatg atgtcttgca ctcaatcaaa ttgaagaatc tgaacaacta catctccctc 240

ttcagaaaga aaacaaggac agaaaaggag aataaggaac ttgaaaattt ggagatcaat 300

ctgaggaaag agatcgcgaa agcctttaaa ggcaacgaag gatacaaaag tctgttcaag 360

aaggatataa ttgagacaat tttgccagag ttcctcgatg acaaggacga gattgcgctg 420

gtcaattcgt tcaacggatt cacaacagca ttcacaggct tctttgataa tcgggaaaat 480

atgttctctg aggaggcaaa gtccacttct attgcgttca ggtgtatcaa tgagaatctc 540

actaggtaca tttccaacat ggatatcttt gagaaggttg acgcaatttt tgacaagcac 600

gaagttcagg agattaagga gaagatcctc aattccgatt atgacgttga ggacttcttc 660

gaaggtgagt tttttaattt cgtgctcact caagagggta tcgacgtgta taatgcgatc 720

atcggtgggt tcgtgactga gtccggtgaa aagattaagg gattgaacga gtatatcaac 780

ctttacaacc aaaagacgaa acagaagctg ccaaagttca agcctcttta caaacaggtt 840

ctttcagacc gcgagtcact ctcgttctat ggggagggct acacttcgga tgaggaagtc 900

ctggaggtgt tcaggaatac tctcaataag aattcggaga ttttctcttc tataaaaaaa 960

ctggaaaagt tgtttaagaa ttttgacgaa tactctagcg ccggcatatt tgtgaaaaac 1020

ggcccggcca tatcaacgat aagtaaagat atcttcggcg aatggaacgt gatcagagac 1080

aaatggaacg cggagtatga cgatattcac ctgaagaaga aggctgtcgt aacggagaag 1140

tacgaggatg atcgcaggaa aagcttcaaa aagatcggaa gtttcagcct ggaacagttg 1200

caggagtatg ctgacgccga tcttagcgtc gtcgagaagt tgaaggagat aatcatccaa 1260

aaggtcgacg agatatataa agtctatgga tcaagtgaaa aactgttcga cgccgacttc 1320

gttttggaga agtccctgaa gaagaacgac gctgttgttg ccattatgaa ggatctgctc 1380

gacagcgtga agagtttcga gaactatatt aaggcttttt tcggggaggg gaaggagact 1440

aacagagatg agtccttcta cggagacttc gtcctcgcgt acgatatact ccttaaggta 1500

gaccacatct acgacgcaat cagaaattac gtgacacaaa agccgtacag caaggacaag 1560

ttcaaactct acttccagaa cccccagttc atgggcggct gggacaagga caaggaaacg 1620

gattacaggg ctacgatcct gaggtatggt tcaaaatact acttggcgat tatggacaag 1680

aagtacgcca agtgtctcca gaagattgac aaagacgatg tcaatggcaa ttatgagaag 1740

atcaactaca agctgcttcc gggtccgaac aagatgctcc caaaggtttt cttcagcaag 1800

aaatggatgg cctactataa cccaagcgag gacatccaga agatttataa gaacggtacg 1860

ttcaagaagg gcgacatgtt caatcttaac gactgtcaca agctgatcga cttcttcaaa 1920

gactcaatta gccggtaccc aaagtggtct aacgcctatg acttcaactt ttcggaaacc 1980

gagaagtaca aggatatagc cggattttat agagaggtgg aagagcaggg ctacaaggtg 2040

tcattcgagt ccgccagcaa gaaggaagtg gacaagctcg tggaagaggg taagctctac 2100

atgttccaga tttataataa agactttagc gataagagcc acgggacacc taatctccac 2160

acaatgtatt tcaagctgct cttcgacgag aataaccacg gccaaatcag gttgtcagga 2220

ggggctgaac tcttcatgcg gcgcgctagc cttaagaagg aggagcttgt agtccaccct 2280

gcgaatagtc caattgcgaa taagaacccg gacaatccta aaaagactac aacattgagc 2340

tacgacgtgt acaaggataa gaggttttcc gaggatcagt acgagctcca catcccgatt 2400

gcgatcaaca agtgcccaaa gaatattttc aagataaaca cagaggtgcg tgtactcctg 2460

aagcatgacg acaatcctta cgtcattggg attgctcggg gcgagaggaa cctcctctat 2520

attgtggtgg tggacgggaa ggggaacata gtcgaacagt actcccttaa cgaaataatt 2580

aacaatttca acggcatccg tatcaagacc gactaccatt cgttgctgga caagaaggag 2640

aaggagagat ttgaggcgcg gcaaaattgg acaagtatcg agaacatcaa ggaactcaaa 2700

gcaggttata tctctcaagt tgtgcataag atatgcgagc tggttgagaa gtatgacgca 2760

gtgatcgctc ttgaggacct caactcgggc tttaagaatt ctagagttaa agtggagaag 2820

caggtctatc aaaagttcga gaagatgctt atagataagc tcaactacat ggtcgataag 2880

aaatcgaacc catgtgccac cggcggcgca ctcaaaggtt accaaataac aaacaaattc 2940

gagtccttca aatcgatgag tactcagaat gggttcatat tttatatacc ggcgtggctt 3000

acgtctaaga tcgacccgtc aactggtttt gtcaacctgt tgaagacgaa atacacgtcc 3060

attgccgatt cgaaaaagtt catatctagt tttgatcgta ttatgtacgt cccagaggaa 3120

gatcttttcg agtttgctct cgactacaaa aacttttcgc ggaccgatgc ggattacatt 3180

aaaaaatgga aactctattc gtacggcaac agaatcagga tttttcgcaa ccctaagaag 3240

aataacgtct ttgattggga ggaagtttgc ttgactagcg cgtacaagga gctctttaat 3300

aagtatggca ttaactacca acagggtgat atcagagcac tgctttgcga acaatctgac 3360

aaggctttct actcatcctt catggctttg atgagcctga tgctccagat gagaaattca 3420

attacaggca gaaccgacgt ggatttcttg atctccccgg ttaaaaattc tgatggcatc 3480

ttttacgata gcaggaacta tgaagcgcaa gagaatgcga ttctgccaaa aaatgcagac 3540

gccaacggtg cctataacat cgccaggaaa gtcctgtggg cgatcggcca gttcaaaaag 3600

gccgaagacg aaaaattgga caaggtcaaa atcgctatca gcaacaaaga gtggctggag 3660

tatgctcaga catccgtaaa gcataagctt atgccaaaaa agaagagaaa ggtttcaggc 3720

ggctcctccg aggtggagtt ctctcacgag tattggatga ggcacgctct tacacttgct 3780

aagagagctt gggacgaaag agaagtgcca gttggcgccg ttcttgtgca taataatagg 3840

gtgatcggcg agggttggaa tagaccaatt ggaaggcatg atccaacagc tcacgcagag 3900

attatggctc tcagacaagg cggcctcgtt atgcagaact acaggctcat tgacgctaca 3960

ctctacgtga cactcgaacc ttgcgttatg tgcgccggag ctatgattca ttctaggatt 4020

ggcagggtcg tgtttggagc tagggacgct aaaacaggag ccgccggatc tcttatggac 4080

gtgttgcatc atccaggcat gaaccatagg gtggagatta cagagggcat tcttgcagac 4140

gagtgcgctg ctcttctttc cgatttcttc aggatgagaa ggcaggagat taaggcccag 4200

aagaaggctc agtcttctac agatagcgga ggatcttccg gaggatctag cggctccgag 4260

acaccaggaa catccgaaag cgctacacca gaatctagcg gaggctcttc cggaggatct 4320

tctgaagtgg agttctccca cgagtattgg atgaggcacg ctcttacact tgctaaaagg 4380

gctagggacg aaagggaagt tccagttgga gctgttctcg tgctcaataa cagggtgatt 4440

ggcgagggtt ggaatagagc cattggactc catgatccaa cagctcacgc agagattatg 4500

gctcttagac aaggcggcct cgttatgcag aattacagac tcatcgacgc cacactctac 4560

gttaccttcg aaccttgcgt tatgtgcgcc ggagctatga tccattctag gattggcagg 4620

gtcgtgttcg gcgttagaaa cgctaagaca ggagctgcag gctctcttat ggacgttctt 4680

cattacccag gcatgaatca tagagtggag atcacagaag gcattcttgc agacgagtgc 4740

gcagctctcc tttgctattt cttcaggatg ccgaggcaag ttttcaacgc tcagaagaag 4800

gcccagtctt ctacagattc cggcggatct tctggaggat ctagcggctc cgagacacca 4860

ggaacatccg aatccgctac accagagtct tctggaggat ctagcggagg atctcttaag 4920

aagagaccag cagctacaaa gaaggccgga caagctaaga agaagaagta g 4971

<210> 15

<211> 4992

<212> DNA

<213> Artificial Sequence

<220>

<223> LBCPF1-ABE2-X3

<400> 15

atgccaaaaa agaagagaaa ggtttcaaag ctcgagaaat tcaccaactg ttattcgttg 60

agcaaaacac tgcggtttaa agcgattcca gtcggcaaga ctcaagagaa tatagacaat 120

aagcggctgt tggtggaaga tgaaaagcgc gcggaagact acaaaggggt gaagaagttg 180

ttggacagat actacctctc ttttatcaat gatgtcttgc actcaatcaa attgaagaat 240

ctgaacaact acatctccct cttcagaaag aaaacaagga cagaaaagga gaataaggaa 300

cttgaaaatt tggagatcaa tctgaggaaa gagatcgcga aagcctttaa aggcaacgaa 360

ggatacaaaa gtctgttcaa gaaggatata attgagacaa ttttgccaga gttcctcgat 420

gacaaggacg agattgcgct ggtcaattcg ttcaacggat tcacaacagc attcacaggc 480

ttctttgata atcgggaaaa tatgttctct gaggaggcaa agtccacttc tattgcgttc 540

aggtgtatca atgagaatct cactaggtac atttccaaca tggatatctt tgagaaggtt 600

gacgcaattt ttgacaagca cgaagttcag gagattaagg agaagatcct caattccgat 660

tatgacgttg aggacttctt cgaaggtgag ttttttaatt tcgtgctcac tcaagagggt 720

atcgacgtgt ataatgcgat catcggtggg ttcgtgactg agtccggtga aaagattaag 780

ggattgaacg agtatatcaa cctttacaac caaaagacga aacagaagct gccaaagttc 840

aagcctcttt acaaacaggt tctttcagac cgcgagtcac tctcgttcta tggggagggc 900

tacacttcgg atgaggaagt cctggaggtg ttcaggaata ctctcaataa gaattcggag 960

attttctctt ctataaaaaa actggaaaag ttgtttaaga attttgacga atactctagc 1020

gccggcatat ttgtgaaaaa cggcccggcc atatcaacga taagtaaaga tatcttcggc 1080

gaatggaacg tgatcagaga caaatggaac gcggagtatg acgatattca cctgaagaag 1140

aaggctgtcg taacggagaa gtacgaggat gatcgcagga aaagcttcaa aaagatcgga 1200

agtttcagcc tggaacagtt gcaggagtat gctgacgccg atcttagcgt cgtcgagaag 1260

ttgaaggaga taatcatcca aaaggtcgac gagatatata aagtctatgg atcaagtgaa 1320

aaactgttcg acgccgactt cgttttggag aagtccctga agaagaacga cgctgttgtt 1380

gccattatga aggatctgct cgacagcgtg aagagtttcg agaactatat taaggctttt 1440

ttcggggagg ggaaggagac taacagagat gagtccttct acggagactt cgtcctcgcg 1500

tacgatatac tccttaaggt agaccacatc tacgacgcaa tcagaaatta cgtgacacaa 1560

aagccgtaca gcaaggacaa gttcaaactc tacttccaga acccccagtt catgggcggc 1620

tgggacaagg acaaggaaac ggattacagg gctacgatcc tgaggtatgg ttcaaaatac 1680

tacttggcga ttatggacaa gaagtacgcc aagtgtctcc agaagattga caaagacgat 1740

gtcaatggca attatgagaa gatcaactac aagctgcttc cgggtccgaa caagatgctc 1800

ccaaaggttt tcttcagcaa gaaatggatg gcctactata acccaagcga ggacatccag 1860

aagatttata agaacggtac gttcaagaag ggcgacatgt tcaatcttaa cgactgtcac 1920

aagctgatcg acttcttcaa agactcaatt agccggtacc caaagtggtc taacgcctat 1980

gacttcaact tttcggaaac cgagaagtac aaggatatag ccggatttta tagagaggtg 2040

gaagagcagg gctacaaggt gtcattcgag tccgccagca agaaggaagt ggacaagctc 2100

gtggaagagg gtaagctcta catgttccag atttataata aagactttag cgataagagc 2160

cacgggacac ctaatctcca cacaatgtat ttcaagctgc tcttcgacga gaataaccac 2220

ggccaaatca ggttgtcagg aggggctgaa ctcttcatgc ggcgcgctag ccttaagaag 2280

gaggagcttg tagtccaccc tgcgaatagt ccaattgcga ataagaaccc ggacaatcct 2340

aaaaagacta caacattgag ctacgacgtg tacaaggata agaggttttc cgaggatcag 2400

tacgagctcc acatcccgat tgcgatcaac aagtgcccaa agaatatttt caagataaac 2460

acagaggtgc gtgtactcct gaagcatgac gacaatcctt acgtcattgg gattgctcgg 2520

ggcgagagga acctcctcta tattgtggtg gtggacggga aggggaacat agtcgaacag 2580

tactccctta acgaaataat taacaatttc aacggcatcc gtatcaagac cgactaccat 2640

tcgttgctgg acaagaagga gaaggagaga tttgaggcgc ggcaaaattg gacaagtatc 2700

gagaacatca aggaactcaa agcaggttat atctctcaag ttgtgcataa gatatgcgag 2760

ctggttgaga agtatgacgc agtgatcgct cttgaggacc tcaactcggg ctttaagaat 2820

tctagagtta aagtggagaa gcaggtctat caaaagttcg agaagatgct tatagataag 2880

ctcaactaca tggtcgataa gaaatcgaac ccatgtgcca ccggcggcgc actcaaaggt 2940

taccaaataa caaacaaatt cgagtccttc aaatcgatga gtactcagaa tgggttcata 3000

ttttatatac cggcgtggct tacgtctaag atcgacccgt caactggttt tgtcaacctg 3060

ttgaagacga aatacacgtc cattgccgat tcgaaaaagt tcatatctag ttttgatcgt 3120

attatgtacg tcccagagga agatcttttc gagtttgctc tcgactacaa aaacttttcg 3180

cggaccgatg cggattacat taaaaaatgg aaactctatt cgtacggcaa cagaatcagg 3240

atttttcgca accctaagaa gaataacgtc tttgattggg aggaagtttg cttgactagc 3300

gcgtacaagg agctctttaa taagtatggc attaactacc aacagggtga tatcagagca 3360

ctgctttgcg aacaatctga caaggctttc tactcatcct tcatggcttt gatgagcctg 3420

atgctccaga tgagaaattc aattacaggc agaaccgacg tggatttctt gatctccccg 3480

gttaaaaatt ctgatggcat cttttacgat agcaggaact atgaagcgca agagaatgcg 3540

attctgccaa aaaatgcaga cgccaacggt gcctataaca tcgccaggaa agtcctgtgg 3600

gcgatcggcc agttcaaaaa ggccgaagac gaaaaattgg acaaggtcaa aatcgctatc 3660

agcaacaaag agtggctgga gtatgctcag acatccgtaa agcataagct tatgccaaaa 3720

aagaagagaa aggtttcagg cggctcctcc gaggtggagt tctctcacga gtattggatg 3780

aggcacgctc ttacacttgc taagagagct tgggacgaaa gagaagtgcc agttggcgcc 3840

gttcttgtgc ataataatag ggtgatcggc gagggttgga atagaccaat tggaaggcat 3900

gatccaacag ctcacgcaga gattatggct ctcagacaag gcggcctcgt tatgcagaac 3960

tacaggctca ttgacgctac actctacgtg acactcgaac cttgcgttat gtgcgccgga 4020

gctatgattc attctaggat tggcagggtc gtgtttggag ctagggacgc taaaacagga 4080

gccgccggat ctcttatgga cgtgttgcat catccaggca tgaaccatag ggtggagatt 4140

acagagggca ttcttgcaga cgagtgcgct gctcttcttt ccgatttctt caggatgaga 4200

aggcaggaga ttaaggccca gaagaaggct cagtcttcta cagatagcgg aggatcttcc 4260

ggaggatcta gcggctccga gacaccagga acatccgaaa gcgctacacc agaatctagc 4320

ggaggctctt ccggaggatc ttctgaagtg gagttctccc acgagtattg gatgaggcac 4380

gctcttacac ttgctaaaag ggctagggac gaaagggaag ttccagttgg agctgttctc 4440

gtgctcaata acagggtgat tggcgagggt tggaatagag ccattggact ccatgatcca 4500

acagctcacg cagagattat ggctcttaga caaggcggcc tcgttatgca gaattacaga 4560

ctcatcgacg ccacactcta cgttaccttc gaaccttgcg ttatgtgcgc cggagctatg 4620

atccattcta ggattggcag ggtcgtgttc ggcgttagaa acgctaagac aggagctgca 4680

ggctctctta tggacgttct tcattaccca ggcatgaatc atagagtgga gatcacagaa 4740

ggcattcttg cagacgagtg cgcagctctc ctttgctatt tcttcaggat gccgaggcaa 4800

gttttcaacg ctcagaagaa ggcccagtct tctacagatt ccggcggatc ttctggagga 4860

tctagcggct ccgagacacc aggaacatcc gaatccgcta caccagagtc ttctggagga 4920

tctagcggag gatctcttaa gaagagacca gcagctacaa agaaggccgg acaagctaag 4980

aagaagaagt ag 4992

<210> 16

<211> 5686

<212> DNA

<213> Artificial Sequence

<220>

<223> PJIT163-GFP

<400> 16

gagctcggta cctgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa 60

gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat 120

ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat 180

atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag 240

tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt 300

tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg 360

tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta 420

gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata atttagatat 480

aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa 540

actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac 600

gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac 660

ggcacggcat ctctgtcgct gcctctggac ccctctcgat cgagagttcc gctccaccgt 720

tggacttgct ccgctgtcgg catccagaaa ttgcgtggcg gagcggcaga cgtgagccgg 780

cacggcaggc ggcctcctcc tcctctcacg gcaccggcag ctacggggga ttcctttccc 840

accgctcctt cgctttccct tcctcgcccg ccgtaataaa tagacacccc ctccacaccc 900

tctttcccca acctcgtgtt gttcggagcg cacacacaca caaccagatc tcccccaaat 960

ccacccgtcg gcacctccgc ttcaaggtac gccgctcgtc ctcccccccc ccccctctct 1020

accttctcta gatcggcgtt ccggtccatg gttagggccc ggtagttcta cttctgttca 1080

tgtttgtgtt agatccgtgt ttgtgttaga tccgtgctgc tagcgttcgt acacggatgc 1140

gacctgtacg tcagacacgt tctgattgct aacttgccag tgtttctctt tggggaatcc 1200

tgggatggct ctagccgttc cgcagacggg atcgatttca tgattttttt tgtttcgttg 1260

catagggttt ggtttgccct tttcctttat ttcaatatat gccgtgcact tgtttgtcgg 1320

gtcatctttt catgcttttt tttgtcttgg ttgtgatgat gtggtctggt tgggcggtcg 1380

ttctagatcg gagtagaatt aattctgttt caaactacct ggtggattta ttaattttgg 1440

atctgtatgt gtgtgccata catattcata gttacgaatt gaagatgatg gatggaaata 1500

tcgatctagg ataggtatac atgttgatgc gggttttact gatgcatata cagagatgct 1560

ttttgttcgc ttggttgtga tgatgtggtg tggttgggcg gtcgttcatt cgttctagat 1620

cggagtagaa tactgtttca aactacctgg tgtatttatt aattttggaa ctgtatgtgt 1680

gtgtcataca tcttcatagt tacgagttta agatggatgg aaatatcgat ctaggatagg 1740

tatacatgtt gatgtgggtt ttactgatgc atatacatga tggcatatgc agcatctatt 1800

catatgctct aaccttgagt acctatctat tataataaac aagtatgttt tataattatt 1860

ttgatcttga tatacttgga tgatggcata tgcagcagct atatgtggat ttttttagcc 1920

ctgccttcat acgctattta tttgcttggt actgtttctt ttgtcgatgc tcaccctgtt 1980

gtttggtgtt acttctgcaa agcttgtcga cggatccatg gtgagcaagg gcgaggagct 2040

gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 2100

cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 2160

ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcacctacgg 2220

cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc 2280

catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 2340

gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 2400

catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 2460

ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 2520

ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 2580

catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct 2640

gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 2700

cgggatcact cacggcatgg acgagctgta caagtaaccc gggaattcgg tacgctgaaa 2760

tcaccagtct ctctctacaa atctatctct ctctattttc tccataaata atgtgtgagt 2820

agtttcccga taagggaaat tagggttctt atagggtttc gctcatgtgt tgagcatata 2880

agaaaccctt agtatgtatt tgtatttgta aaatacttct atcaataaaa tttctaattc 2940

ctaaaaccaa aatccagtac taaaatccag atctcctaaa gtccctatag atctttgtcg 3000

tgaatataaa ccagacacga gacgactaaa cctggagccc agacgccgtt cgaagctaga 3060

agtaccgctt aggcaggagg ccgttaggga aaagatgcta aggcagggtt ggttacgttg 3120

actcccccgt aggtttggtt taaatatgat gaagtggacg gaaggaagga ggaagacaag 3180

gaaggataag gttgcaggcc ctgtgcaagg taagaagatg gaaatttgat agaggtacgc 3240

tactatactt atactatacg ctaagggaat gcttgtattt ataccctata ccccctaata 3300

accccttatc aatttaagaa ataatccgca taagcccccg cttaaaaatt ggtatcagag 3360

ccatgaatag gtctatgacc aaaactcaag aggataaaac ctcaccaaaa tacgaaagag 3420

ttcttaactc taaagataaa agatctttca agatcaaaac tagttccctc acaccggagc 3480

atgcgatatc ctcgagagat ctaggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 3540

tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 3600

ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 3660

tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 3720

ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 3780

ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 3840

gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 3900

gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 3960

cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 4020

ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 4080

tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 4140

gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 4200

tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 4260

ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 4320

ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 4380

ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 4440

accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 4500

tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 4560

cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 4620

taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 4680

caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 4740

gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 4800

gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 4860

ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 4920

attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 4980

gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 5040

tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 5100

agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 5160

gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 5220

actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 5280

tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 5340

attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 5400

tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 5460

tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 5520

aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 5580

tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 5640

cgcacatttc cccgaaaagt gccacctgcc agtgccaagc taattc 5686

<210> 17

<211> 720

<212> DNA

<213> Artificial Sequence

<220>

<223> pBUI-mGFP

<400> 17

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccttcaccta cggcgtgtag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actcacggca tggacgagct gtacaagtaa 720

<210> 18

<211> 1307

<212> PRT

<213> Acidaminococcus sp.

<400> 18

Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln

20 25 30

Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys

35 40 45

Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln

50 55 60

Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile

65 70 75 80

Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile

85 90 95

Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly

100 105 110

Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile

115 120 125

Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys

130 135 140

Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg

145 150 155 160

Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg

165 170 175

Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg

180 185 190

Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe

195 200 205

Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn

210 215 220

Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val

225 230 235 240

Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp

245 250 255

Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu

260 265 270

Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn

275 280 285

Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro

290 295 300

Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu

305 310 315 320

Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr

325 330 335

Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu

340 345 350

Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His

355 360 365

Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr

370 375 380

Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys

385 390 395 400

Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu

405 410 415

Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser

420 425 430

Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala

435 440 445

Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys

450 455 460

Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu

465 470 475 480

Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe

485 490 495

Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser

500 505 510

Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val

515 520 525

Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp

530 535 540

Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn

545 550 555 560

Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys

565 570 575

Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys

580 585 590

Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys

595 600 605

Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr

610 615 620

Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys

625 630 635 640

Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln

645 650 655

Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala

660 665 670

Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr

675 680 685

Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr

690 695 700

Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His

705 710 715 720

Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu

725 730 735

Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys

740 745 750

Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu

755 760 765

Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln

770 775 780

Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His

785 790 795 800

Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr

805 810 815

Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His

820 825 830

Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn

835 840 845

Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe

850 855 860

Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln

865 870 875 880

Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu

885 890 895

Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg

900 905 910

Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu

915 920 925

Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu

930 935 940

Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val

945 950 955 960

Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile

965 970 975

His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu

980 985 990

Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu

995 1000 1005

Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu

1010 1015 1020

Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly

1025 1030 1035

Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala

1040 1045 1050

Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro

1055 1060 1065

Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe

1070 1075 1080

Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu

1085 1090 1095

Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe

1100 1105 1110

Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly

1115 1120 1125

Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn

1130 1135 1140

Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys

1145 1150 1155

Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr

1160 1165 1170

Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu

1175 1180 1185

Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu

1190 1195 1200

Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu

1205 1210 1215

Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly

1220 1225 1230

Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys

1235 1240 1245

Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp

1250 1255 1260

Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu

1265 1270 1275

Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile

1280 1285 1290

Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn

1295 1300 1305

<210> 19

<211> 1300

<212> PRT

<213> Francisella novicida

<400> 19

Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys

20 25 30

Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys

35 40 45

Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu

50 55 60

Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser

65 70 75 80

Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys

85 90 95

Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr

100 105 110

Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile

115 120 125

Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln

130 135 140

Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr

145 150 155 160

Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr

165 170 175

Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

180 185 190

Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

195 200 205

Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys

210 215 220

Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

225 230 235 240

Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg

245 250 255

Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr

260 265 270

Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys

275 280 285

Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile

290 295 300

Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys

305 310 315 320

Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

325 330 335

Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met

340 345 350

Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys

355 360 365

Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln

370 375 380

Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr

385 390 395 400

Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala

405 410 415

Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn

420 425 430

Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala

435 440 445

Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn

450 455 460

Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala

465 470 475 480

Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys

485 490 495

Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys

500 505 510

Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp

515 520 525

Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His

530 535 540

Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His

545 550 555 560

Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val

565 570 575

Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser

580 585 590

Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly

595 600 605

Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys

610 615 620

Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile

625 630 635 640

Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys

645 650 655

Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val

660 665 670

Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile

675 680 685

Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

690 695 700

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe

705 710 715 720

Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp

725 730 735

Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu

740 745 750

Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn

755 760 765

Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr

770 775 780

Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg

785 790 795 800

Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

805 810 815

Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr

820 825 830

Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

835 840 845

Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu

850 855 860

Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe

865 870 875 880

His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe

885 890 895

Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His

900 905 910

Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu

915 920 925

Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

930 935 940

Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile

945 950 955 960

Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

965 970 975

Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile

980 985 990

Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu

995 1000 1005

Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val

1010 1015 1020

Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu

1025 1030 1035

Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg

1040 1045 1050

Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly

1055 1060 1065

Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser

1070 1075 1080

Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys

1085 1090 1095

Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp

1100 1105 1110

Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe

1115 1120 1125

Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr

1130 1135 1140

Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp

1145 1150 1155

Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu

1160 1165 1170

Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly

1175 1180 1185

Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe

1190 1195 1200

Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg

1205 1210 1215

Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val

1220 1225 1230

Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys

1235 1240 1245

Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly

1250 1255 1260

Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu

1265 1270 1275

Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu

1280 1285 1290

Phe Val Gln Asn Arg Asn Asn

1295 1300

<210> 20

<211> 1228

<212> PRT

<213> Lachnospiraceae bacterium

<400> 20

Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp

20 25 30

Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys

35 40 45

Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp

50 55 60

Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu

65 70 75 80

Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn

85 90 95

Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn

100 105 110

Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu

115 120 125

Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe

130 135 140

Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn

145 150 155 160

Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile

165 170 175

Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys

180 185 190

Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys

195 200 205

Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe

210 215 220

Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile

225 230 235 240

Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn

245 250 255

Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys

260 265 270

Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser

275 280 285

Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe

290 295 300

Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys

305 310 315 320

Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile

325 330 335

Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe

340 345 350

Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp

355 360 365

Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp

370 375 380

Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu

385 390 395 400

Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu

405 410 415

Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser

420 425 430

Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys

435 440 445

Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys

450 455 460

Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr

465 470 475 480

Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile

485 490 495

Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr

500 505 510

Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro

515 520 525

Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala

530 535 540

Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys

545 550 555 560

Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly

565 570 575

Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met

580 585 590

Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro

595 600 605

Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly

610 615 620

Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys

625 630 635 640

Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn

645 650 655

Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu

660 665 670

Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys

675 680 685

Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile

690 695 700

Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His

705 710 715 720

Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile

725 730 735

Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys

740 745 750

Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys

755 760 765

Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr

770 775 780

Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile

785 790 795 800

Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val

805 810 815

Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp

820 825 830

Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly

835 840 845

Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn

850 855 860

Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu

865 870 875 880

Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile

885 890 895

Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys

900 905 910

Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn

915 920 925

Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln

930 935 940

Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys

945 950 955 960

Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile

965 970 975

Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe

980 985 990

Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr

995 1000 1005

Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp

1010 1015 1020

Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro

1025 1030 1035

Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser

1040 1045 1050

Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr

1055 1060 1065

Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val

1070 1075 1080

Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu

1085 1090 1095

Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala

1100 1105 1110

Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met

1115 1120 1125

Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly

1130 1135 1140

Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp

1145 1150 1155

Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala

1160 1165 1170

Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala

1175 1180 1185

Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp

1190 1195 1200

Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp

1205 1210 1215

Leu Glu Tyr Ala Gln Thr Ser Val Lys His

1220 1225

<210> 21

<211> 1329

<212> PRT

<213> Artificial Sequence

<220>

<223> ASCPF1-2NLS

<400> 21

Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln

20 25 30

Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys

35 40 45

Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln

50 55 60

Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile

65 70 75 80

Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile

85 90 95

Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly

100 105 110

Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile

115 120 125

Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys

130 135 140

Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg

145 150 155 160

Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg

165 170 175

Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg

180 185 190

Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe

195 200 205

Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn

210 215 220

Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val

225 230 235 240

Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp

245 250 255

Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu

260 265 270

Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn

275 280 285

Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro

290 295 300

Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu

305 310 315 320

Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr

325 330 335

Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu

340 345 350

Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His

355 360 365

Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr

370 375 380

Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys

385 390 395 400

Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu

405 410 415

Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser

420 425 430

Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala

435 440 445

Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys

450 455 460

Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu

465 470 475 480

Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe

485 490 495

Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser

500 505 510

Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val

515 520 525

Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp

530 535 540

Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn

545 550 555 560

Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys

565 570 575

Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys

580 585 590

Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys

595 600 605

Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr

610 615 620

Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys

625 630 635 640

Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln

645 650 655

Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala

660 665 670

Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr

675 680 685

Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr

690 695 700

Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His

705 710 715 720

Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu

725 730 735

Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys

740 745 750

Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu

755 760 765

Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln

770 775 780

Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His

785 790 795 800

Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr

805 810 815

Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His

820 825 830

Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn

835 840 845

Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe

850 855 860

Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln

865 870 875 880

Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu

885 890 895

Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg

900 905 910

Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu

915 920 925

Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu

930 935 940

Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val

945 950 955 960

Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile

965 970 975

His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu

980 985 990

Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu

995 1000 1005

Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu

1010 1015 1020

Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly

1025 1030 1035

Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala

1040 1045 1050

Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro

1055 1060 1065

Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe

1070 1075 1080

Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu

1085 1090 1095

Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe

1100 1105 1110

Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly

1115 1120 1125

Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn

1130 1135 1140

Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys

1145 1150 1155

Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr

1160 1165 1170

Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu

1175 1180 1185

Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu

1190 1195 1200

Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu

1205 1210 1215

Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly

1220 1225 1230

Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys

1235 1240 1245

Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp

1250 1255 1260

Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu

1265 1270 1275

Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile

1280 1285 1290

Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn Ser

1295 1300 1305

Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Pro

1310 1315 1320

Lys Lys Lys Arg Lys Val

1325

<210> 22

<211> 1322

<212> PRT

<213> Artificial Sequence

<220>

<223> FNCPF1-2NLS

<400> 22

Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys

20 25 30

Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys

35 40 45

Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu

50 55 60

Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser

65 70 75 80

Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys

85 90 95

Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr

100 105 110

Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile

115 120 125

Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln

130 135 140

Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr

145 150 155 160

Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr

165 170 175

Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

180 185 190

Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

195 200 205

Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys

210 215 220

Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

225 230 235 240

Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg

245 250 255

Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr

260 265 270

Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys

275 280 285

Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile

290 295 300

Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys

305 310 315 320

Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

325 330 335

Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met

340 345 350

Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys

355 360 365

Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln

370 375 380

Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr

385 390 395 400

Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala

405 410 415

Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn

420 425 430

Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala

435 440 445

Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn

450 455 460

Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala

465 470 475 480

Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys

485 490 495

Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys

500 505 510

Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp

515 520 525

Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His

530 535 540

Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His

545 550 555 560

Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val

565 570 575

Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser

580 585 590

Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly

595 600 605

Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys

610 615 620

Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile

625 630 635 640

Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys

645 650 655

Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val

660 665 670

Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile

675 680 685

Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

690 695 700

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe

705 710 715 720

Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp

725 730 735

Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu

740 745 750

Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn

755 760 765

Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr

770 775 780

Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg

785 790 795 800

Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

805 810 815

Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr

820 825 830

Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

835 840 845

Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu

850 855 860

Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe

865 870 875 880

His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe

885 890 895

Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His

900 905 910

Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu

915 920 925

Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

930 935 940

Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile

945 950 955 960

Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

965 970 975

Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile

980 985 990

Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu

995 1000 1005

Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val

1010 1015 1020

Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu

1025 1030 1035

Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg

1040 1045 1050

Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly

1055 1060 1065

Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser

1070 1075 1080

Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys

1085 1090 1095

Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp

1100 1105 1110

Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe

1115 1120 1125

Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr

1130 1135 1140

Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp

1145 1150 1155

Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu

1160 1165 1170

Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly

1175 1180 1185

Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe

1190 1195 1200

Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg

1205 1210 1215

Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val

1220 1225 1230

Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys

1235 1240 1245

Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly

1250 1255 1260

Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu

1265 1270 1275

Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu

1280 1285 1290

Phe Val Gln Asn Arg Asn Asn Ser Gly Gly Ser Pro Lys Lys Lys

1295 1300 1305

Arg Lys Val Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val

1310 1315 1320

<210> 23

<211> 1252

<212> PRT

<213> Artificial Sequence

<220>

<223> LBCPF1-2NLS

<400> 23

Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp

20 25 30

Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys

35 40 45

Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp

50 55 60

Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu

65 70 75 80

Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn

85 90 95

Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn

100 105 110

Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu

115 120 125

Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe

130 135 140

Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn

145 150 155 160

Met Glu Thr Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg

165 170 175

Cys Ile Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe

180 185 190

Glu Lys Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys

195 200 205

Glu Lys Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly

210 215 220

Glu Phe Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn

225 230 235 240

Ala Ile Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly

245 250 255

Leu Asn Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu

260 265 270

Pro Lys Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser

275 280 285

Leu Ser Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu

290 295 300

Val Phe Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile

305 310 315 320

Lys Lys Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala

325 330 335

Gly Ile Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp

340 345 350

Ile Phe Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr

355 360 365

Asp Asp Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu

370 375 380

Asp Asp Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu

385 390 395 400

Gln Leu Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu

405 410 415

Lys Glu Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly

420 425 430

Ser Ser Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu

435 440 445

Lys Lys Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser

450 455 460

Val Lys Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys

465 470 475 480

Glu Thr Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr

485 490 495

Asp Ile Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr

500 505 510

Val Thr Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln

515 520 525

Asn Pro Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr

530 535 540

Arg Ala Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met

545 550 555 560

Asp Lys Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val

565 570 575

Asn Gly Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn

580 585 590

Lys Met Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr

595 600 605

Asn Pro Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys

610 615 620

Lys Gly Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe

625 630 635 640

Phe Lys Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp

645 650 655

Phe Asn Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr

660 665 670

Arg Glu Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser

675 680 685

Lys Lys Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe

690 695 700

Gln Ile Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn

705 710 715 720

Leu His Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly

725 730 735

Gln Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser

740 745 750

Leu Lys Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala

755 760 765

Asn Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp

770 775 780

Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile

785 790 795 800

Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr

805 810 815

Glu Val Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly

820 825 830

Ile Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly

835 840 845

Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn

850 855 860

Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys

865 870 875 880

Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu

885 890 895

Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys

900 905 910

Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp

915 920 925

Leu Asn Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val

930 935 940

Tyr Gln Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val

945 950 955 960

Asp Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr

965 970 975

Gln Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn

980 985 990

Gly Phe Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro

995 1000 1005

Ser Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile

1010 1015 1020

Ala Asp Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr

1025 1030 1035

Val Pro Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn

1040 1045 1050

Phe Ser Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr

1055 1060 1065

Ser Tyr Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn

1070 1075 1080

Asn Val Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys

1085 1090 1095

Glu Leu Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile

1100 1105 1110

Arg Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser

1115 1120 1125

Phe Met Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile

1130 1135 1140

Thr Gly Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn

1145 1150 1155

Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu

1160 1165 1170

Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn

1175 1180 1185

Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala

1190 1195 1200

Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys

1205 1210 1215

Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His Ser Gly Gly

1220 1225 1230

Ser Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Pro Lys Lys

1235 1240 1245

Lys Arg Lys Val

1250

<210> 24

<211> 1664

<212> PRT

<213> Artificial Sequence

<220>

<223> dFNCPF1-PBE-2NLS

<400> 24

Met Pro Lys Lys Lys Arg Lys Val Ser Ser Glu Thr Gly Pro Val Ala

1 5 10 15

Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val

20 25 30

Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu

35 40 45

Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln Asn

50 55 60

Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu

65 70 75 80

Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser

85 90 95

Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser

100 105 110

Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His

115 120 125

His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser

130 135 140

Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp

145 150 155 160

Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg

165 170 175

Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile

180 185 190

Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro

195 200 205

Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg

210 215 220

Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Ser Gly Ser Glu

225 230 235 240

Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Leu Lys Ser Ile Tyr

245 250 255

Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Glu

260 265 270

Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly Leu

275 280 285

Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys Gln

290 295 300

Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser Ser

305 310 315 320

Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr Phe

325 330 335

Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys Ser

340 345 350

Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp Ser

355 360 365

Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys Lys

370 375 380

Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp Asn

385 390 395 400

Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp Glu

405 410 415

Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe Lys

420 425 430

Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile Pro

435 440 445

Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe Leu

450 455 460

Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu Ala

465 470 475 480

Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr Phe

485 490 495

Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser Leu

500 505 510

Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln Ser

515 520 525

Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn Gly

530 535 540

Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr Ser

545 550 555 560

Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val Leu

565 570 575

Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile Asp

580 585 590

Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe Tyr

595 600 605

Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys Glu

610 615 620

Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp Leu

625 630 635 640

Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser Gln

645 650 655

Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu Tyr

660 665 670

Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys Lys

675 680 685

Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu Ser

690 695 700

Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg Asp

705 710 715 720

Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala Ala

725 730 735

Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu Ala

740 745 750

Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu Gln

755 760 765

Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp Gln

770 775 780

Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln Ser

785 790 795 800

Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu Val

805 810 815

Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr Asn

820 825 830

Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys Phe

835 840 845

Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys Asn

850 855 860

Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys Tyr

865 870 875 880

Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp Lys

885 890 895

Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr Lys

900 905 910

Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser Ala

915 920 925

Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile Arg

930 935 940

Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr Glu

945 950 955 960

Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe Tyr

965 970 975

Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe Arg

980 985 990

Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg Glu

995 1000 1005

Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu

1010 1015 1020

Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr Leu Phe

1025 1030 1035

Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg Pro

1040 1045 1050

Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

1055 1060 1065

Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe

1070 1075 1080

Tyr Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys

1085 1090 1095

Glu Ala Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser

1100 1105 1110

Val Phe Glu Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp

1115 1120 1125

Lys Phe Phe Phe His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser

1130 1135 1140

Gly Ala Asn Lys Phe Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu

1145 1150 1155

Lys Ala Asn Asp Val His Ile Leu Ser Ile Ala Arg Gly Glu Arg

1160 1165 1170

His Leu Ala Tyr Tyr Thr Leu Val Asp Gly Lys Gly Asn Ile Ile

1175 1180 1185

Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp Arg Met Lys Thr

1190 1195 1200

Asn Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp Arg Asp Ser

1205 1210 1215

Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu Met Lys

1220 1225 1230

Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile Ala Lys Leu Val

1235 1240 1245

Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly

1250 1255 1260

Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys

1265 1270 1275

Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe Lys

1280 1285 1290

Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln

1295 1300 1305

Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr

1310 1315 1320

Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys

1325 1330 1335

Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser

1340 1345 1350

Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp Lys Ile Cys

1355 1360 1365

Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe Asp Tyr Lys

1370 1375 1380

Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr Ile Ala Ser

1385 1390 1395

Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp Lys Asn His

1400 1405 1410

Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu Leu Glu Lys

1415 1420 1425

Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly Glu Cys Ile

1430 1435 1440

Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe Phe Ala Lys

1445 1450 1455

Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn Ser Lys

1460 1465 1470

Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp Val

1475 1480 1485

Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro

1490 1495 1500

Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys Gly

1505 1510 1515

Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys Lys

1520 1525 1530

Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln

1535 1540 1545

Asn Arg Asn Asn Thr Arg Asp Ser Gly Gly Ser Thr Asn Leu Ser

1550 1555 1560

Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu

1565 1570 1575

Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn

1580 1585 1590

Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser

1595 1600 1605

Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr

1610 1615 1620

Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys

1625 1630 1635

Ile Lys Met Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val

1640 1645 1650

Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val

1655 1660

<210> 25

<211> 1592

<212> PRT

<213> Artificial Sequence

<220>

<223> dLBCPF1-PBE-2NLS

<400> 25

Met Pro Lys Lys Lys Arg Lys Val Ser Ser Glu Thr Gly Pro Val Ala

1 5 10 15

Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val

20 25 30

Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu

35 40 45

Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln Asn

50 55 60

Thr Asn Lys His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu

65 70 75 80

Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser

85 90 95

Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser

100 105 110

Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His

115 120 125

His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser

130 135 140

Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp

145 150 155 160

Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg

165 170 175

Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile

180 185 190

Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro

195 200 205

Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg

210 215 220

Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu Lys Ser Gly Ser Glu

225 230 235 240

Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Leu Lys Ser Lys Leu

245 250 255

Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys

260 265 270

Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu

275 280 285

Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys

290 295 300

Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu His Ser

305 310 315 320

Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys

325 330 335

Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn

340 345 350

Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys

355 360 365

Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu

370 375 380

Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe Asn Gly Phe Thr

385 390 395 400

Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn Met Phe Ser Glu

405 410 415

Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu

420 425 430

Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys Val Asp Ala Ile

435 440 445

Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser

450 455 460

Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe Val

465 470 475 480

Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe

485 490 495

Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn

500 505 510

Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu

515 520 525

Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu

530 535 540

Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe Arg Asn Thr Leu

545 550 555 560

Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu

565 570 575

Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn

580 585 590

Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn

595 600 605

Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile His Leu Lys

610 615 620

Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser

625 630 635 640

Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala

645 650 655

Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu Ile Ile Ile Gln

660 665 670

Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu Phe

675 680 685

Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys Asn Asp Ala Val

690 695 700

Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys Ser Phe Glu Asn

705 710 715 720

Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu

725 730 735

Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys Val

740 745 750

Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr Gln Lys Pro Tyr

755 760 765

Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly

770 775 780

Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr Ile Leu Arg

785 790 795 800

Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys

805 810 815

Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly Asn Tyr Glu Lys

820 825 830

Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val

835 840 845

Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile

850 855 860

Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp Met Phe Asn

865 870 875 880

Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser

885 890 895

Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu Thr

900 905 910

Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu Glu Gln

915 920 925

Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys Glu Val Asp Lys

930 935 940

Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr Asn Lys Asp

945 950 955 960

Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His Thr Met Tyr Phe

965 970 975

Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile Arg Leu Ser Gly

980 985 990

Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys Lys Glu Glu Leu

995 1000 1005

Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn Pro Asp

1010 1015 1020

Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys Asp

1025 1030 1035

Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile Ala

1040 1045 1050

Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val

1055 1060 1065

Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile

1070 1075 1080

Ala Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly

1085 1090 1095

Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn

1100 1105 1110

Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu

1115 1120 1125

Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr

1130 1135 1140

Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln

1145 1150 1155

Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala Val

1160 1165 1170

Ile Ala Leu Glu Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg Val

1175 1180 1185

Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile

1190 1195 1200

Asp Lys Leu Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys Ala

1205 1210 1215

Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe Glu

1220 1225 1230

Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr Ile

1235 1240 1245

Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe Val

1250 1255 1260

Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys

1265 1270 1275

Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu Asp

1280 1285 1290

Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp

1295 1300 1305

Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg

1310 1315 1320

Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp

1325 1330 1335

Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys

1340 1345 1350

Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys

1355 1360 1365

Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu Met

1370 1375 1380

Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly Arg Thr Asp

1385 1390 1395

Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp Gly Ile Phe

1400 1405 1410

Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala Ile Leu Pro

1415 1420 1425

Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Val

1430 1435 1440

Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp Glu Lys Leu

1445 1450 1455

Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp Leu Glu Tyr

1460 1465 1470

Ala Gln Thr Ser Val Lys His Thr Arg Asp Ser Gly Gly Ser Thr

1475 1480 1485

Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val

1490 1495 1500

Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val

1505 1510 1515

Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr

1520 1525 1530

Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala

1535 1540 1545

Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly

1550 1555 1560

Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Pro Lys Lys Lys

1565 1570 1575

Arg Lys Val Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val

1580 1585 1590

<210> 26

<211> 1731

<212> PRT

<213> Artificial Sequence

<220>

<223> dFNCPF1-ABE7.10-2NLS

<400> 26

Met Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Ser Glu Val Glu

1 5 10 15

Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg

20 25 30

Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn

35 40 45

Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp

50 55 60

Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val

65 70 75 80

Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu

85 90 95

Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg

100 105 110

Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu

115 120 125

Met Asp Val Leu His His Pro Gly Met Asn His Arg Val Glu Ile Thr

130 135 140

Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe

145 150 155 160

Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys Ala Gln Ser Ser

165 170 175

Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro

180 185 190

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly

195 200 205

Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala

210 215 220

Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly

225 230 235 240

Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg

245 250 255

Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu

260 265 270

Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr

275 280 285

Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile

290 295 300

His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr

305 310 315 320

Gly Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn

325 330 335

His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala

340 345 350

Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln

355 360 365

Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser

370 375 380

Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser

385 390 395 400

Ser Gly Gly Ser Ser Gly Gly Ser Leu Lys Ser Ile Tyr Gln Glu Phe

405 410 415

Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Glu Leu Ile Pro

420 425 430

Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly Leu Ile Leu Asp

435 440 445

Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys Gln Ile Ile Asp

450 455 460

Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser Ser Val Cys Ile

465 470 475 480

Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr Phe Lys Leu Lys

485 490 495

Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys Ser Ala Lys Asp

500 505 510

Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp Ser Glu Lys Phe

515 520 525

Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys Lys Gly Gln Glu

530 535 540

Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp Asn Gly Ile Glu

545 550 555 560

Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp Glu Ala Leu Glu

565 570 575

Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe Lys Gly Phe His

580 585 590

Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile Pro Thr Ser Ile

595 600 605

Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe Leu Glu Asn Lys

610 615 620

Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu Ala Ile Asn Tyr

625 630 635 640

Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr Phe Asp Ile Asp

645 650 655

Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser Leu Asp Glu Val

660 665 670

Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln Ser Gly Ile Thr

675 680 685

Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn Gly Glu Asn Thr

690 695 700

Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr Ser Gln Gln Ile

705 710 715 720

Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val Leu Phe Lys Gln

725 730 735

Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile Asp Lys Leu Glu

740 745 750

Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe Tyr Glu Gln Ile

755 760 765

Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys Glu Thr Leu Ser

770 775 780

Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp Leu Ser Lys Ile

785 790 795 800

Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser Gln Gln Val Phe

805 810 815

Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu Tyr Ile Thr Gln

820 825 830

Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys Lys Glu Gln Glu

835 840 845

Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu Ser Leu Glu Thr

850 855 860

Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg Asp Ile Asp Lys

865 870 875 880

Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala Ala Ile Pro Met

885 890 895

Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu Ala Gln Ile Ser

900 905 910

Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu Gln Ala Ser Ala

915 920 925

Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp Gln Thr Asn Asn

930 935 940

Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln Ser Glu Asp Lys

945 950 955 960

Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu Val Phe Glu Glu

965 970 975

Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr Asn Lys Ile Arg

980 985 990

Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys Phe Lys Leu Asn

995 1000 1005

Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys Asn Lys Glu

1010 1015 1020

Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys Tyr Tyr

1025 1030 1035

Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp Lys

1040 1045 1050

Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr

1055 1060 1065

Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe

1070 1075 1080

Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu

1085 1090 1095

Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

1100 1105 1110

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys

1115 1120 1125

Phe Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp

1130 1135 1140

Lys Asp Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser

1145 1150 1155

Ile Asp Glu Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu

1160 1165 1170

Thr Phe Glu Asn Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn

1175 1180 1185

Gln Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser

1190 1195 1200

Ala Tyr Ser Lys Gly Arg Pro Asn Leu His Thr Leu Tyr Trp Lys

1205 1210 1215

Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp Val Val Tyr Lys Leu

1220 1225 1230

Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln Ser Ile Pro Lys

1235 1240 1245

Lys Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn Lys Asn Lys

1250 1255 1260

Asp Asn Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu Ile Lys

1265 1270 1275

Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro Ile

1280 1285 1290

Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu

1295 1300 1305

Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His Ile Leu

1310 1315 1320

Ser Ile Ala Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu Val

1325 1330 1335

Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

1340 1345 1350

Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala

1355 1360 1365

Ile Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile

1370 1375 1380

Asn Asn Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val

1385 1390 1395

His Glu Ile Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val

1400 1405 1410

Phe Glu Asp Leu Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val

1415 1420 1425

Glu Lys Gln Val Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys

1430 1435 1440

Leu Asn Tyr Leu Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly

1445 1450 1455

Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe

1460 1465 1470

Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala

1475 1480 1485

Gly Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln

1490 1495 1500

Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe

1505 1510 1515

Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe

1520 1525 1530

Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys

1535 1540 1545

Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe

1550 1555 1560

Arg Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr

1565 1570 1575

Pro Thr Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu

1580 1585 1590

Tyr Gly His Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser

1595 1600 1605

Asp Lys Lys Phe Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile

1610 1615 1620

Leu Gln Met Arg Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu

1625 1630 1635

Ile Ser Pro Val Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg

1640 1645 1650

Gln Ala Pro Lys Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala

1655 1660 1665

Tyr His Ile Gly Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys

1670 1675 1680

Asn Asn Gln Glu Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu

1685 1690 1695

Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn Asn Ser Gly Gly Ser

1700 1705 1710

Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Pro Lys Lys Lys

1715 1720 1725

Arg Lys Val

1730

<210> 27

<211> 1659

<212> PRT

<213> Artificial Sequence

<220>

<223> dLBCPF1-ABE7.10-2NLS

<400> 27

Met Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Ser Glu Val Glu

1 5 10 15

Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg

20 25 30

Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn

35 40 45

Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp

50 55 60

Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val

65 70 75 80

Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu

85 90 95

Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg

100 105 110

Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu

115 120 125

Met Asp Val Leu His His Pro Gly Met Asn His Arg Val Glu Ile Thr

130 135 140

Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe

145 150 155 160

Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys Ala Gln Ser Ser

165 170 175

Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro

180 185 190

Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly

195 200 205

Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala

210 215 220

Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly

225 230 235 240

Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg

245 250 255

Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu

260 265 270

Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr

275 280 285

Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile

290 295 300

His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr

305 310 315 320

Gly Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn

325 330 335

His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala

340 345 350

Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln

355 360 365

Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser

370 375 380

Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser

385 390 395 400

Ser Gly Gly Ser Ser Gly Gly Ser Leu Lys Ser Lys Leu Glu Lys Phe

405 410 415

Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys Ala Ile Pro

420 425 430

Val Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu Leu Val Glu

435 440 445

Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys Leu Leu Asp

450 455 460

Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu His Ser Ile Lys Leu

465 470 475 480

Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys Thr Arg Thr

485 490 495

Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn Leu Arg Lys

500 505 510

Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys Ser Leu Phe

515 520 525

Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu Asp Asp Lys

530 535 540

Asp Glu Ile Ala Leu Val Asn Ser Phe Asn Gly Phe Thr Thr Ala Phe

545 550 555 560

Thr Gly Phe Phe Asp Asn Arg Glu Asn Met Phe Ser Glu Glu Ala Lys

565 570 575

Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu Thr Arg Tyr

580 585 590

Ile Ser Asn Met Asp Ile Phe Glu Lys Val Asp Ala Ile Phe Asp Lys

595 600 605

His Glu Val Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser Asp Tyr Asp

610 615 620

Val Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe Val Leu Thr Gln

625 630 635 640

Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe Val Thr Glu

645 650 655

Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Asn

660 665 670

Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu Tyr Lys Gln

675 680 685

Val Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly Tyr Thr

690 695 700

Ser Asp Glu Glu Val Leu Glu Val Phe Arg Asn Thr Leu Asn Lys Asn

705 710 715 720

Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu Phe Lys Asn

725 730 735

Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn Gly Pro Ala

740 745 750

Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn Val Ile Arg

755 760 765

Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile His Leu Lys Lys Lys Ala

770 775 780

Val Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser Phe Lys Lys

785 790 795 800

Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala Asp Ala Asp

805 810 815

Leu Ser Val Val Glu Lys Leu Lys Glu Ile Ile Ile Gln Lys Val Asp

820 825 830

Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu Phe Asp Ala Asp

835 840 845

Phe Val Leu Glu Lys Ser Leu Lys Lys Asn Asp Ala Val Val Ala Ile

850 855 860

Met Lys Asp Leu Leu Asp Ser Val Lys Ser Phe Glu Asn Tyr Ile Lys

865 870 875 880

Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu Ser Phe Tyr

885 890 895

Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys Val Asp His Ile

900 905 910

Tyr Asp Ala Ile Arg Asn Tyr Val Thr Gln Lys Pro Tyr Ser Lys Asp

915 920 925

Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly Gly Trp Asp

930 935 940

Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr Ile Leu Arg Tyr Gly Ser

945 950 955 960

Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys Cys Leu Gln

965 970 975

Lys Ile Asp Lys Asp Asp Val Asn Gly Asn Tyr Glu Lys Ile Asn Tyr

980 985 990

Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val Phe Phe Ser

995 1000 1005

Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile Gln Lys

1010 1015 1020

Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp Met Phe Asn Leu

1025 1030 1035

Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser

1040 1045 1050

Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu

1055 1060 1065

Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu

1070 1075 1080

Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys Glu

1085 1090 1095

Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile

1100 1105 1110

Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu

1115 1120 1125

His Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly

1130 1135 1140

Gln Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala

1145 1150 1155

Ser Leu Lys Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro

1160 1165 1170

Ile Ala Asn Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu

1175 1180 1185

Ser Tyr Asp Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr

1190 1195 1200

Glu Leu His Ile Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile

1205 1210 1215

Phe Lys Ile Asn Thr Glu Val Arg Val Leu Leu Lys His Asp Asp

1220 1225 1230

Asn Pro Tyr Val Ile Gly Ile Ala Arg Gly Glu Arg Asn Leu Leu

1235 1240 1245

Tyr Ile Val Val Val Asp Gly Lys Gly Asn Ile Val Glu Gln Tyr

1250 1255 1260

Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn Gly Ile Arg Ile Lys

1265 1270 1275

Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe

1280 1285 1290

Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile Lys Glu Leu

1295 1300 1305

Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys Glu Leu

1310 1315 1320

Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn Ser

1325 1330 1335

Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln

1340 1345 1350

Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp

1355 1360 1365

Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr

1370 1375 1380

Gln Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln

1385 1390 1395

Asn Gly Phe Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile

1400 1405 1410

Asp Pro Ser Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr

1415 1420 1425

Ser Ile Ala Asp Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile

1430 1435 1440

Met Tyr Val Pro Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr

1445 1450 1455

Lys Asn Phe Ser Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys

1460 1465 1470

Leu Tyr Ser Tyr Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys

1475 1480 1485

Lys Asn Asn Val Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala

1490 1495 1500

Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly

1505 1510 1515

Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr

1520 1525 1530

Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn

1535 1540 1545

Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val

1550 1555 1560

Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala

1565 1570 1575

Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala

1580 1585 1590

Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys

1595 1600 1605

Lys Ala Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser

1610 1615 1620

Asn Lys Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His Ser

1625 1630 1635

Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Gly Gly Ser Pro

1640 1645 1650

Lys Lys Lys Arg Lys Val

1655

<210> 28

<211> 1656

<212> PRT

<213> Artificial Sequence

<220>

<223> LBCPF1-ABE2-X2

<400> 28

Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp

20 25 30

Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys

35 40 45

Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp

50 55 60

Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu

65 70 75 80

Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn

85 90 95

Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn

100 105 110

Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu

115 120 125

Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe

130 135 140

Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn

145 150 155 160

Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile

165 170 175

Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys

180 185 190

Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys

195 200 205

Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe

210 215 220

Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile

225 230 235 240

Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn

245 250 255

Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys

260 265 270

Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser

275 280 285

Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe

290 295 300

Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys

305 310 315 320

Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile

325 330 335

Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe

340 345 350

Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp

355 360 365

Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp

370 375 380

Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu

385 390 395 400

Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu

405 410 415

Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser

420 425 430

Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys

435 440 445

Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys

450 455 460

Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr

465 470 475 480

Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile

485 490 495

Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr

500 505 510

Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro

515 520 525

Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala

530 535 540

Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys

545 550 555 560

Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly

565 570 575

Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met

580 585 590

Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro

595 600 605

Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly

610 615 620

Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys

625 630 635 640

Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn

645 650 655

Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu

660 665 670

Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys

675 680 685

Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile

690 695 700

Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His

705 710 715 720

Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile

725 730 735

Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys

740 745 750

Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys

755 760 765

Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr

770 775 780

Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile

785 790 795 800

Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val

805 810 815

Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Ala

820 825 830

Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly

835 840 845

Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn

850 855 860

Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu

865 870 875 880

Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile

885 890 895

Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys

900 905 910

Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn

915 920 925

Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln

930 935 940

Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys

945 950 955 960

Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile

965 970 975

Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe

980 985 990

Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr

995 1000 1005

Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp

1010 1015 1020

Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro

1025 1030 1035

Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser

1040 1045 1050

Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr

1055 1060 1065

Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val

1070 1075 1080

Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu

1085 1090 1095

Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala

1100 1105 1110

Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met

1115 1120 1125

Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly

1130 1135 1140

Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp

1145 1150 1155

Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala

1160 1165 1170

Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala

1175 1180 1185

Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp

1190 1195 1200

Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp

1205 1210 1215

Leu Glu Tyr Ala Gln Thr Ser Val Lys His Lys Leu Met Pro Lys

1220 1225 1230

Lys Lys Arg Lys Val Ser Gly Gly Ser Ser Glu Val Glu Phe Ser

1235 1240 1245

His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala

1250 1255 1260

Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn

1265 1270 1275

Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His

1280 1285 1290

Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly

1295 1300 1305

Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val

1310 1315 1320

Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser

1325 1330 1335

Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly

1340 1345 1350

Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn

1355 1360 1365

His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala

1370 1375 1380

Ala Leu Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys

1385 1390 1395

Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser

1400 1405 1410

Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala

1415 1420 1425

Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Glu Val

1430 1435 1440

Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala

1445 1450 1455

Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu

1460 1465 1470

Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile

1475 1480 1485

Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

1490 1495 1500

Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr

1505 1510 1515

Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met

1520 1525 1530

Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala

1535 1540 1545

Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro

1550 1555 1560

Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp

1565 1570 1575

Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln

1580 1585 1590

Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly

1595 1600 1605

Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser

1610 1615 1620

Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser

1625 1630 1635

Leu Lys Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys

1640 1645 1650

Lys Lys Lys

1655

<210> 29

<211> 1663

<212> PRT

<213> Artificial Sequence

<220>

<223> LBCPF1-ABE2-X3

<400> 29

Met Pro Lys Lys Lys Arg Lys Val Ser Lys Leu Glu Lys Phe Thr Asn

1 5 10 15

Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys Ala Ile Pro Val Gly

20 25 30

Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu Leu Val Glu Asp Glu

35 40 45

Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys Leu Leu Asp Arg Tyr

50 55 60

Tyr Leu Ser Phe Ile Asn Asp Val Leu His Ser Ile Lys Leu Lys Asn

65 70 75 80

Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys Thr Arg Thr Glu Lys

85 90 95

Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn Leu Arg Lys Glu Ile

100 105 110

Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys Ser Leu Phe Lys Lys

115 120 125

Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu Asp Asp Lys Asp Glu

130 135 140

Ile Ala Leu Val Asn Ser Phe Asn Gly Phe Thr Thr Ala Phe Thr Gly

145 150 155 160

Phe Phe Asp Asn Arg Glu Asn Met Phe Ser Glu Glu Ala Lys Ser Thr

165 170 175

Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu Thr Arg Tyr Ile Ser

180 185 190

Asn Met Asp Ile Phe Glu Lys Val Asp Ala Ile Phe Asp Lys His Glu

195 200 205

Val Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser Asp Tyr Asp Val Glu

210 215 220

Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe Val Leu Thr Gln Glu Gly

225 230 235 240

Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe Val Thr Glu Ser Gly

245 250 255

Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Asn Gln Lys

260 265 270

Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu Tyr Lys Gln Val Leu

275 280 285

Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly Tyr Thr Ser Asp

290 295 300

Glu Glu Val Leu Glu Val Phe Arg Asn Thr Leu Asn Lys Asn Ser Glu

305 310 315 320

Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu Phe Lys Asn Phe Asp

325 330 335

Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn Gly Pro Ala Ile Ser

340 345 350

Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn Val Ile Arg Asp Lys

355 360 365

Trp Asn Ala Glu Tyr Asp Asp Ile His Leu Lys Lys Lys Ala Val Val

370 375 380

Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser Phe Lys Lys Ile Gly

385 390 395 400

Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala Asp Ala Asp Leu Ser

405 410 415

Val Val Glu Lys Leu Lys Glu Ile Ile Ile Gln Lys Val Asp Glu Ile

420 425 430

Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu Phe Asp Ala Asp Phe Val

435 440 445

Leu Glu Lys Ser Leu Lys Lys Asn Asp Ala Val Val Ala Ile Met Lys

450 455 460

Asp Leu Leu Asp Ser Val Lys Ser Phe Glu Asn Tyr Ile Lys Ala Phe

465 470 475 480

Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu Ser Phe Tyr Gly Asp

485 490 495

Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys Val Asp His Ile Tyr Asp

500 505 510

Ala Ile Arg Asn Tyr Val Thr Gln Lys Pro Tyr Ser Lys Asp Lys Phe

515 520 525

Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly Gly Trp Asp Lys Asp

530 535 540

Lys Glu Thr Asp Tyr Arg Ala Thr Ile Leu Arg Tyr Gly Ser Lys Tyr

545 550 555 560

Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys Cys Leu Gln Lys Ile

565 570 575

Asp Lys Asp Asp Val Asn Gly Asn Tyr Glu Lys Ile Asn Tyr Lys Leu

580 585 590

Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val Phe Phe Ser Lys Lys

595 600 605

Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile Gln Lys Ile Tyr Lys

610 615 620

Asn Gly Thr Phe Lys Lys Gly Asp Met Phe Asn Leu Asn Asp Cys His

625 630 635 640

Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser Arg Tyr Pro Lys Trp

645 650 655

Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu Thr Glu Lys Tyr Lys Asp

660 665 670

Ile Ala Gly Phe Tyr Arg Glu Val Glu Glu Gln Gly Tyr Lys Val Ser

675 680 685

Phe Glu Ser Ala Ser Lys Lys Glu Val Asp Lys Leu Val Glu Glu Gly

690 695 700

Lys Leu Tyr Met Phe Gln Ile Tyr Asn Lys Asp Phe Ser Asp Lys Ser

705 710 715 720

His Gly Thr Pro Asn Leu His Thr Met Tyr Phe Lys Leu Leu Phe Asp

725 730 735

Glu Asn Asn His Gly Gln Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe

740 745 750

Met Arg Arg Ala Ser Leu Lys Lys Glu Glu Leu Val Val His Pro Ala

755 760 765

Asn Ser Pro Ile Ala Asn Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr

770 775 780

Thr Leu Ser Tyr Asp Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln

785 790 795 800

Tyr Glu Leu His Ile Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile

805 810 815

Phe Lys Ile Asn Thr Glu Val Arg Val Leu Leu Lys His Asp Asp Asn

820 825 830

Pro Tyr Val Ile Gly Ile Ala Arg Gly Glu Arg Asn Leu Leu Tyr Ile

835 840 845

Val Val Val Asp Gly Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn

850 855 860

Glu Ile Ile Asn Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr His

865 870 875 880

Ser Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn

885 890 895

Trp Thr Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser

900 905 910

Gln Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala Val

915 920 925

Ile Ala Leu Glu Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg Val Lys

930 935 940

Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile Asp Lys

945 950 955 960

Leu Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly

965 970 975

Ala Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser

980 985 990

Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr Ile Pro Ala Trp Leu Thr

995 1000 1005

Ser Lys Ile Asp Pro Ser Thr Gly Phe Val Asn Leu Leu Lys Thr

1010 1015 1020

Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys Phe Ile Ser Ser Phe

1025 1030 1035

Asp Arg Ile Met Tyr Val Pro Glu Glu Asp Leu Phe Glu Phe Ala

1040 1045 1050

Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp Ala Asp Tyr Ile Lys

1055 1060 1065

Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg Ile Arg Ile Phe Arg

1070 1075 1080

Asn Pro Lys Lys Asn Asn Val Phe Asp Trp Glu Glu Val Cys Leu

1085 1090 1095

Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile Asn Tyr

1100 1105 1110

Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp Lys

1115 1120 1125

Ala Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln

1130 1135 1140

Met Arg Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile

1145 1150 1155

Ser Pro Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn

1160 1165 1170

Tyr Glu Ala Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala

1175 1180 1185

Asn Gly Ala Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly

1190 1195 1200

Gln Phe Lys Lys Ala Glu Asp Glu Lys Leu Asp Lys Val Lys Ile

1205 1210 1215

Ala Ile Ser Asn Lys Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val

1220 1225 1230

Lys His Lys Leu Met Pro Lys Lys Lys Arg Lys Val Ser Gly Gly

1235 1240 1245

Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala

1250 1255 1260

Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val

1265 1270 1275

Gly Ala Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp

1280 1285 1290

Asn Arg Pro Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile

1295 1300 1305

Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu

1310 1315 1320

Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu Pro Cys Val Met Cys

1325 1330 1335

Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly

1340 1345 1350

Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val

1355 1360 1365

Leu His His Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly

1370 1375 1380

Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe Arg

1385 1390 1395

Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys Ala Gln Ser Ser

1400 1405 1410

Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr

1415 1420 1425

Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser

1430 1435 1440

Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met

1445 1450 1455

Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu

1460 1465 1470

Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly

1475 1480 1485

Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His

1490 1495 1500

Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn

1505 1510 1515

Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys

1520 1525 1530

Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val

1535 1540 1545

Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu

1550 1555 1560

Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg Val Glu Ile

1565 1570 1575

Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys Tyr

1580 1585 1590

Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys Ala

1595 1600 1605

Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly

1610 1615 1620

Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

1625 1630 1635

Gly Gly Ser Ser Gly Gly Ser Leu Lys Lys Arg Pro Ala Ala Thr

1640 1645 1650

Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1655 1660

<210> 30

<211> 1526

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial Sequence

<400> 30

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

tcgacgagtc taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag 660

cagacggcac ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg 720

ttggacttgc tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg 780

gcacggcagg cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc 840

caccgctcct tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc 900

ctctttcccc aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa 960

tccacccgtc ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc 1020

taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc 1080

atgtttgtgt tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg 1140

cgacctgtac gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc 1200

ctgggatggc tctagccgtt ccgcagacgg gatcgatcta ggataggtat acatgttgat 1260

gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 1320

cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 1380

acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 1440

ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 1500

tctgcaggtc gaagcttgaa gcaaac 1526

Claims (8)

1. A system for base editing a target sequence in a plant genome, comprising at least one of the following i) to v):

i) Base-editing fusion proteins, and guide RNAs;

ii) an expression construct comprising a nucleotide sequence encoding a base-editing fusion protein, and a guide RNA;

iii) A base-editing fusion protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;

iv) an expression construct comprising a nucleotide sequence encoding a base-editing fusion protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;

v) an expression construct comprising a nucleotide sequence encoding a base-editing fusion protein and a nucleotide sequence encoding a guide RNA;

wherein the base-editing fusion protein comprises Cpf1 lacking DNA cleavage activity and a deaminase, the guide RNA being capable of targeting the base-editing fusion protein to a target sequence in a genome resulting in a substitution of one or more of C to T or A to G in the target sequence,

wherein the Cpf1 with the DNA cleavage activity deletion is FnCpf1 with the DNA cleavage activity deletion or LbCpf1 with the DNA cleavage activity deletion,

wherein the base-editing fusion protein further comprises 3 Nuclear Localization Sequences (NLS), wherein 1 is located at the N-terminus of the base-editing fusion protein and 2 is located at the C-terminus of the base-editing fusion protein,

wherein the plant is wheat or rice,

wherein the amino acid sequence of the FnCpf1 base editing fusion protein containing DNA cutting activity deletion is shown as SEQ ID NO. 24 or SEQ ID NO. 26, and the amino acid sequence of the LbCpf1 base editing fusion protein containing DNA cutting activity deletion is shown as SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 28 or SEQ ID NO. 29.

2. The system of claim 1, wherein the nucleotide sequence encoding the base-editing fusion protein is codon optimized for an organism to be base-edited.

3. The system of claim 1, wherein the nucleotide sequence encoding the base-editing fusion protein and/or the nucleotide sequence encoding the guide RNA is operably linked to an expression control element.

4. The system of claim 3, wherein the regulatory element is a promoter.

5. The system of claim 4, wherein the promoter is a 35S promoter, a maize Ubi-1 promoter, a wheat U6 promoter, a rice U3 promoter, or a maize U3 promoter.

6. A method of producing a genetically modified plant comprising introducing the system of any one of claims 1-5 into a cell of an organism, whereby the guide RNA targets the base-editing fusion protein to a target sequence in the genome of the cell, resulting in one or more C to T or a to G substitutions in the target sequence.

7. The method of claim 6, wherein the plant comprises a monocot and a dicot.

8. The method of claim 6 or 7, wherein the plant is rice or wheat.

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