CN114703203B

CN114703203B - Baculovirus vector and use thereof

Info

Publication number: CN114703203B
Application number: CN202210127524.1A
Authority: CN
Inventors: 潘雨堃; 王天天
Original assignee: Shanghai Boyin Biotechnology Co ltd
Current assignee: Shanghai Boyin Biotechnology Co ltd
Priority date: 2022-02-11
Filing date: 2022-02-11
Publication date: 2024-08-06
Anticipated expiration: 2042-02-11
Also published as: CN114703203A

Abstract

The present application relates to the preparation of a linear double-stranded endless DNA (neDNA) expression vector comprising AAV Inverted Terminal Repeats (ITRs) and a gene expression cassette using an insect cell-baculovirus system. The insect cell-baculovirus system of the application has high yield of neDNA, and the yield is improved by 2-3 times on average. Compared with other Bac-Rep, the expression stability of Rep protein (Rep 78) is better after 3 continuous baculovirus passages.

Description

Baculovirus vector and use thereof

Technical Field

The application relates to the field of biological medicine, in particular to a baculovirus vector and application thereof.

Background

Gene therapy is a novel therapeutic approach to achieve the goal of treating disease at the nucleic acid level by regulating the biological process of gene expression. The ideal gene therapy vector needs to have the characteristics of high delivery efficiency, high safety, scalable production and low cost. The recombinant adeno-associated virus (rAAV) vector has the characteristics of high efficiency and safety, and is a main gene delivery vector for genetic disease gene therapy at present. rAAV still faces the problems of difficult production and scale-up and high production cost. Non-viral vector gene therapy based on DNA therapy is expected to solve this problem. In 2013, robert Kotin et al prepared a linear double-stranded, end-free DNA (no end DNA, neDNA) closed at both ends by the Inverted Terminal Repeats (ITRs) of the AAV genome using the insect cell baculovirus expression system. The preparation method of Robert Kotin is directly transformed from the AAV baculovirus production system developed in the laboratory 2002, and is not optimized for neDNA in terms of yield; meanwhile, when the recombinant baculovirus Bac-Rep expresses Rep protein, homologous recombination can occur between Rep78 and Rep52, and the system is unstable.

There is thus still a need to overcome the above-mentioned serious limitations of large-scale (commercial) production neDNA in insect cells. It is therefore an object of the present invention to provide means and methods for stable and high-yield (large-scale) production neDNA in insect cells.

Disclosure of Invention

The application aims to design a high-efficiency baculovirus expression system based on insect cells and a method for preparing neDNA by using the baculovirus expression system, optimize a Rep protein expression vector, and improve the stability of Rep protein expression, so that the yield of neDNA and the stability of a production system are improved. The application is characterized in that: 1) The expression level of Rep78 is improved by using a strong promoter (p 10 promoter); 2) The sequence codon of Rep52 is optimized, and homologous recombination of Rep78 and Rep52 sequences is avoided. The application has the following effects: 1) Compared with the preparation method of Robert Kotin, the yield is improved by 2-3 times; 2) Rep protein baculovirus expression vector is stable after 3 generations.

In one aspect, the application provides an isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO. 12.

In certain embodiments, the isolated nucleic acid molecule encodes an adeno-associated virus (AAV) Rep52 protein.

In another aspect, the application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the adeno-associated virus (AAV) Rep78 protein and a nucleotide sequence encoding a Rep52 protein, wherein the nucleic acid molecule encoding a Rep52 protein comprises the nucleotide sequence shown in SEQ ID No. 12.

In certain embodiments, wherein the nucleotide sequence encoding the Rep78 protein is wild-type.

In certain embodiments, wherein the nucleic acid molecule encoding a Rep78 protein comprises the nucleotide sequence set forth in SEQ ID NO. 11.

In certain embodiments, it further comprises a first promoter that initiates transcription of the nucleotide sequence encoding the AAV Rep78 protein and a second promoter that initiates transcription of the nucleic acid molecule encoding the AAV Rep52 protein, the first promoter being the same as or different from the second promoter.

In certain embodiments, the first promoter and the second promoter comprise insect cell promoters.

In certain embodiments, the first promoter comprises a strong promoter.

In certain embodiments, the first promoter has the same or higher transcriptional initiation ability as the second promoter.

In certain embodiments, wherein the first promoter and the second promoter are each independently selected from the group consisting of: p10 promoter, polyhedrin (polh) promoter and IE1 promoter.

In certain embodiments, wherein the transcription direction of the first promoter and the second promoter is the same or opposite.

In certain embodiments, wherein the first promoter is operably linked to the nucleotide sequence encoding the Rep78 protein and the second promoter is operably linked to the nucleotide sequence encoding the Rep52 protein.

In certain embodiments, when the direction of transcription of the first and second promoters is the same, they comprise, in order, the first promoter, the nucleotide sequence encoding the Rep78 protein, the second promoter, and the nucleotide sequence encoding the Rep52 protein.

In certain embodiments, wherein the nucleotide sequence encoding the Rep78 protein and the nucleotide sequence encoding the Rep52 protein further comprise a nucleotide sequence encoding a polyA (pA), respectively.

In certain embodiments, when the transcription direction of the first promoter and the second promoter is the same, it comprises, in order, the first promoter, the nucleotide sequence encoding the Rep78 protein, the first pA, the second promoter, the nucleotide sequence encoding the Rep52 protein, and the second pA.

In certain embodiments, when the transcription directions of the first promoter and the second promoter are opposite, they sequentially comprise a nucleotide sequence encoding Rep78, a first promoter that initiates transcription of the nucleotide sequence encoding Rep78 protein, a second promoter that initiates transcription of the nucleotide sequence encoding Rep52 protein, and a nucleotide sequence encoding Rep 52.

In certain embodiments, wherein the 5 'end of the first promoter is directly or indirectly linked to the 5' end of the second promoter.

In certain embodiments, wherein the 3 'end of the first promoter is directly or indirectly linked to the 5' end of the nucleotide sequence encoding Rep 78.

In certain embodiments, it further comprises a first pA, wherein the 3 'end of the nucleotide sequence encoding the Rep78 protein is directly or indirectly linked to the 5' end of the first pA.

In certain embodiments, wherein the 3 'end of the second promoter is directly or indirectly linked to the 5' end of the nucleotide sequence encoding Rep 52.

In certain embodiments, it further comprises a second pA, wherein the 3 'end of the nucleotide sequence encoding the Rep52 protein is directly or indirectly linked to the 5' end of the second pA.

In certain embodiments, wherein the pA is selected from the group consisting of: any one of SV40 polyA and HSV TK polyA.

In certain embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO. 8.

In another aspect, the application provides an isolated nucleic acid molecule comprising, in order, a first polyA (pA), a nucleotide sequence encoding a Rep78 protein, a first promoter, a second promoter, a protein nucleotide sequence encoding a Rep52 protein, and a second polyA (pA), wherein the first promoter is a nucleotide sequence encoding a Rep78 protein and a transcriptional promoter of the first pA, and the second promoter is a nucleotide sequence encoding a Rep52 protein and a transcriptional promoter of the second polyA, wherein the nucleotide sequence encoding a Rep52 protein and/or the nucleotide sequence encoding a Rep78 protein is codon-optimized to avoid homologous recombination, the first promoter and the second promoter comprising insect cell promoters, and the first promoter is a strong promoter.

In certain embodiments, wherein the first promoter comprises a p10 promoter, a polh promoter, or an IE1 promoter.

In certain embodiments, wherein the p10 promoter comprises the nucleotide sequence set forth in SEQ ID NO. 9.

In certain embodiments, wherein the second promoter comprises a p10 promoter, a polh promoter, or an IE1 promoter.

In certain embodiments, wherein the polh promoter comprises the nucleotide sequence set forth in SEQ ID NO. 10.

In another aspect, the application provides a vector comprising an isolated nucleic acid molecule of the application.

In certain embodiments, the vector comprises a viral vector.

In certain embodiments, the vector comprises a baculovirus vector.

In certain embodiments, the vector comprises a pFastBac vector.

In certain embodiments, the vector comprises the nucleotide sequence set forth in SEQ ID NO. 14.

In another aspect, the application provides a cell comprising an isolated nucleic acid molecule of the application or a vector of the application.

In certain embodiments, the cells comprise insect cells.

In certain embodiments, the cells comprise Spodoptera frugiperda (Sf 9) cells.

In another aspect, the application provides a baculovirus expression system comprising a first baculovirus vector and a second baculovirus vector comprising a nucleic acid sequence encoding a gene of interest, said first baculovirus vector being a baculovirus vector according to the application.

In certain embodiments, the nucleic acid sequence encoding the gene of interest comprises, from 5 'to 3', in order, an Inverted Terminal Repeat (ITR) of the first parvovirus, the gene of interest, and a second ITR.

In certain embodiments, wherein the first ITR further comprises at least one promoter between the first ITR and the gene of interest.

In certain embodiments, wherein the first ITR further comprises at least one eukaryotic promoter between the first ITR and the gene of interest.

In certain embodiments, wherein the first ITR further comprises at least one mammalian cell promoter between the first ITR and the gene of interest.

In certain embodiments, wherein the first ITR further comprises a mammalian cell promoter and an insect cell promoter between the first ITR and the gene of interest.

In certain embodiments, the mammalian cell promoter includes a broad range promoter and a tissue specific promoter.

In certain embodiments, the broad-spectrum promoter comprises a CMV, SV40, EF1a, CAG, or UBC promoter.

In certain embodiments, the tissue-specific promoter comprises an ALB, hAAT, TBG, TTR, GFAP, MHCK7, or hSyn promoter.

In certain embodiments, wherein the mammalian cell promoter comprises a CMV promoter.

In certain embodiments, wherein the insect cell promoter comprises a p10 promoter.

In certain embodiments, the promoter comprises a CMV and p10 promoter.

In another aspect, the application provides an insect cell comprising a first nucleotide sequence encoding a first amino acid sequence and a second nucleotide sequence encoding a second amino acid sequence, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a Rep78 protein and the second nucleotide sequence encodes a Rep52 protein, wherein the first nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO. 11 and the second nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO. 12.

In certain embodiments, wherein the first and second nucleotide sequences are part of a nucleic acid construct, wherein each of the first and second nucleotide sequences is operably linked to an expression control sequence for expression in an insect cell.

In certain embodiments, wherein the insect cell further comprises: a third nucleic acid sequence comprising at least one parvoviral inverted terminal repeat nucleotide sequence (ITR).

In some embodiments, wherein the third nucleotide sequence further comprises at least one nucleotide sequence encoding a gene of interest.

In certain embodiments, wherein the third nucleotide sequence comprises two parvoviral ITR nucleotide sequences, and wherein the at least one nucleotide sequence encoding a gene of interest is located between the two parvoviral ITR nucleotide sequences.

In certain embodiments, wherein the parvovirus comprises an adeno-associated virus.

In certain embodiments, wherein the third nucleotide sequence is part of another nucleic acid construct, wherein each of the nucleotide sequences encoding the gene of interest is operably linked to an expression control sequence for mammalian expression.

In certain embodiments, the nucleic acid construct is an insect cell compatible vector.

In certain embodiments, the nucleic acid construct is a baculovirus vector.

In certain embodiments, it comprises a baculovirus vector described herein.

In another aspect, the application provides the use of a baculovirus expression system of the application or an insect cell of the application in the preparation of a nucleic acid molecule of interest.

In certain embodiments, wherein the nucleic acid molecule of interest is a linear DNA molecule (neDNA) having a covalently closed end.

In another aspect, the application provides a method for producing a nucleic acid molecule of interest comprising culturing an insect cell according to the application.

In certain embodiments, the method of making comprises:

1) Providing a baculovirus expression system according to the present application;

2) Inserting a gene sequence of interest into said second baculovirus vector;

3) Co-transfecting the first baculovirus vector and the second baculovirus vector into an insect cell;

4) Growing the insect cell under conditions that allow replication and release of DNA comprising the gene of interest;

5) Collecting the nucleic acid molecule of interest.

In certain embodiments, the methods further protect against isolation of the nucleic acid molecule of interest.

In another aspect, the application provides a kit comprising an isolated nucleic acid molecule of the application, a baculovirus expression system of the application and/or an insect cell of the application.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the application as claimed. Accordingly, the drawings and descriptions of the present application are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the application related to the application are shown in the appended claims. A better understanding of the features and advantages of the application in accordance with the present application will be obtained by reference to the exemplary embodiments and the accompanying drawings that are described in detail below. The drawings are briefly described as follows:

FIG. 1A shows a plasmid map of the pFastBac-ITR-EGFP of the present application.

FIG. 1B shows a pFastBac-p10Rep plasmid map of the application.

FIG. 2 shows a schematic representation of the transcription of the p10Rep, repWT, inRep and CORep genes according to the present application.

FIG. 3 shows the results of Western Blotting analysis of Rep protein expression and stability; wherein, ctr: uninfected Sf9 cells; 1: bacV-RepWT infected Sf9 cells; 2: bacV-inRep infected Sf9 cells; 3: bacV-CORep infected Sf9 cells; 4: bacV-p10Rep infected Sf9 cells.

FIG. 4 shows the results of the identification of the expression vectors of the neDNA-ITR-EGFP gene driven by different Rep proteins according to the present application; wherein M: DNA MARKER;1-4: electropherograms of RepWT, inRep, CORep and p10 Rep-driven neDNA-ITR-EGFP gene expression vectors, respectively.

FIGS. 5A-5B show the results of the enzymatic cleavage assay of neDNA-ITR-EGFP gene expression vectors of the present application.

FIG. 6 shows fluorescent expression patterns of RepWT, inRep, CORep and p10Rep of the application driving neDNA-ITR-EGFP gene expression vectors, respectively, transfected into HEK293 cells, taken 72h after transfection under a fluorescent microscope.

FIG. 7 shows the results of nanolipid particle delivery neDNA-ITR-FLuc luciferase expression in C57BL/6 mice.

FIG. 8 shows the alignment of Rep52 before and after optimization according to the application (Query: rep52WT, sbjct: rep 52-CO).

Detailed Description

Further advantages and effects of the present application will become readily apparent to those skilled in the art from the present disclosure, by describing embodiments of the present application with specific examples.

Definition of terms

The virus of the parvoviridae family is a small DNA animal virus. Parvoviridae can be divided into two subfamilies: subfamily parvovirus infecting vertebrates and subfamily concha virus infecting insects. Members of the parvoviridae subfamily are referred to herein as parvoviruses and include dependoviruses. As can be inferred from their generic names, the virus-dependent members are unique in that they typically need to co-infect with helper viruses such as adenoviruses or herpesviruses that produce infection in cell culture. Dependoviruses include AAV, which is very common in humans and other primates, and several serotypes have been isolated from various tissue samples. Serotypes 2,3, 5 and 6 were found in human cells, AAV serotypes 1, 4 and 7-11.Kenneth I.Berns,"Parvoviridae：TheViruses and Their Replication,"Chapter 69in Fields Virology(3dEd.1996) in non-human primate samples describe other information about parvoviruses and other members of the parvoviridae family. It is to be understood that the present invention is not limited to AAV, but is equally applicable to other parvoviruses.

The genome of AAV is a linear single stranded DNA molecule less than about 5000 nucleotides (nt) in length. The Inverted Terminal Repeats (ITRs) flank unique coding nucleotide sequences that encode non-structural replication (rep) proteins and structural proteins (VP). The VP proteins (VP 1, VP2 and VP 3) constitute the capsid. The terminal 145nt is self-complementary and ordered so that an energetically stable T-hairpin-forming intramolecular duplex can be formed. These hairpin structures function as origins of viral DNA replication and serve as primers for cellular DNA polymerase complexes. After wtAAV infection of mammalian cells, the Rep genes (i.e., rep78 and Rep 52) are expressed from the P5 promoter and the P19 promoter, respectively, and both expressed Rep proteins play a role in replication of the viral genome. The splicing event in this Rep ORF actually results in the expression of four Rep proteins (i.e., rep78, rep68, rep52, and Rep 40). However, the unspliced mRNA encoding Rep78 and Rep52 is sufficient to produce AAV vectors in mammalian cells. Rep78 and Rep52 proteins are also sufficient to produce AAV vectors in insect cells.

In the present application, the term "AAV vector" or "rAAV vector" generally refers to a vector comprising one or more polynucleotide sequences of interest, genes of interest, or "transgenes" flanked by parvoviruses or AAV Inverted Terminal Repeats (ITRs).

In the present application, the term "operably linked" refers to the linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

In the present application, the term "expression control sequence" generally refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence operably linked thereto. An expression control sequence is "operably linked" to a nucleotide sequence when it controls and regulates the transcription and/or translation of that nucleotide sequence. Thus, a stretch of expression control sequences may include a promoter, an enhancer, an Internal Ribosome Entry Site (IRES), a transcription terminator, a start codon preceding the protein-encoding gene, an intron splice signal, and a stop codon. The term "expression control sequence" is intended to include, at a minimum, a sequence that is present to affect expression, and may include other advantageous components as well. For example, the leader sequence and fusion partner sequence (fusion partner sequence) are expression control sequences. The term may also include nucleic acid sequence designs in which an in-frame and out-of-frame unwanted possible initiation codon is removed from the sequence. It may also include nucleic acid sequence design that removes unwanted possible splice sites. It includes a sequence that directs the addition of a polyA tail, i.e., a string of adenine residues at the 3' end of the mRNA, or polyadenylation sequence (pA), known as a polyA sequence. It can also be designed to increase mRNA stability. Expression control sequences such as promoters and sequences that effect translation such as Kozak sequences are known to exist in insect cells to affect transcription and translation stability. Expression control sequences have the property of modulating the nucleotide sequence to which they are operably linked to thereby reduce or increase the level of expression.

In the present application, the term "promoter" or "transcription regulatory sequence" generally refers to a nucleic acid fragment that has the effect of controlling transcription of one or more coding sequences, which is located upstream of the transcription initiation site of the coding sequence, and which can be recognized structurally by the presence of a DNA-dependent RNA polymerase binding site, a transcription initiation site and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites and any other nucleotide sequence known to those skilled in the art that can directly or indirectly function to regulate the transcription amount of a promoter. A "constitutive" promoter is one that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by the use of chemical inducers. "tissue-specific" promoters are active only in specific tissues or cell types.

In the present application, the term "strong promoter" generally refers to a promoter having a high affinity for RNA polymerase, which is capable of directing synthesis of a large amount of mRNA, i.e., a promoter capable of efficiently promoting transcription of DNA. Mammalian strong promoters include CMV promoter, CAG promoter, EF1a promoter, SV40 promoter, and the like. Insect cell strong promoters include the p10 promoter, polh promoter, IE1 promoter, and the like. Taking the insect cell-baculovirus expression system p10 promoter as an example in the specification: the p10 promoter (p 10 promoter) is a p10 promoter derived from late expression in the alfalfa silver vein moth nucleopolyhedrovirus (AcMNPV), and is a sequence that can drive the high expression of a gene of interest in insect cells.

In the present application, the term "linear double-stranded end-free DNA (nendna)" generally refers to a linear, double-stranded, closed-end DNA vector, both ends of which are closed by the Inverted Terminal Repeats (ITRs) of the AAV genome. neDNA vectors have covalent blocking and are therefore resistant to exonucleases such as exonuclease I or exonuclease III. neDNA can have a variety of configurations, such as: monomers, dimers, trimers, and multimers, and the like.

In the present application, the term "vector" or "construct" is generally a nucleic acid molecule (typically DNA or RNA) for transferring a passenger nucleic acid sequence (i.e., DNA or RNA) to a host cell. Three common types of vectors include plasmids, phages and viruses. The vector is preferably a virus. Vectors containing both a promoter and a cloning site into which a polynucleotide may be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from suppliers such as Stratagene (La Jolla, calif.) and PromegaBiotech (Madison, ffis.). To optimize expression and/or in vitro transcription, it is desirable to remove, add, or alter the 5 'and/or 3' untranslated portions of the clone to remove additional variable translation initiation codons that may be inappropriate or other sequences that interfere with or reduce expression at the transcriptional or translational level.

In the present application, the term "viral vector" generally refers to viral genes, control sequences and viral packaging sequences comprising some or all of the following vector-encoding gene products.

In the present application, a "parvoviral vector" may be defined as a recombinantly produced parvovirus or parvoviral particle comprising a polynucleotide to be delivered into a host cell (in vivo, ex vivo, or in vitro). Examples of parvoviral vectors include, for example, adeno-associated viral vectors.

In the present application, the term "baculovirus insect cell expression system" or "BEVS" (Baculovirus expression vector system) generally refers to a eukaryotic expression system that expresses a foreign protein. The most widely used baculovirus expression system is the split virus Autographa california multiply enveloped nuclear polyhedrosisvirus (AcMNPV), abbreviated as baculovirus (baculovirus). The vector is characterized in that a baculovirus expression system totally uses protein modification, processing and transportation systems existing in higher eukaryotic cells, and belongs to eukaryotic expression systems; acMNPV is a non-helper virus, and can be suitable for mass proliferation in insect cells growing in suspension without any auxiliary factors, so that a large amount of recombinant proteins can be conveniently expressed; the expression system makes the expression product in a dissolved state; the baculovirus gene is large (130 kb) and is suitable for cloning large fragment exogenous genes. Baculovirus-non-infectious vertebrate promoters are inactive in mammalian cells.

In the present application, the term "Bacmid" or "Bacmid" generally refers to recombinant DNA of Baculovirus that is capable of shuttling between insect cells and e.

In the present application, the terms "substantially identical", "substantially identical" or "substantially similar" generally refer to two peptide sequences or two nucleotide sequences that share at least a certain percentage of sequence identity when optimally aligned, e.g., by the GAP or BESTFIT procedure using default parameters, as defined elsewhere in the specification. GAP uses Needleman and Wunsch full sequence alignment algorithm to align two full length sequences and maximize the number of matches and minimize the number of GAPs. GAP default parameters are typically used with a GAP creation penalty = 50 (nucleotides)/8 (proteins) and a GAP extension penalty = 3 (nucleotides)/2 (proteins). The default scoring matrix used for nucleotides was nwsgapdna and for proteins was Blosum62 (Henikoff & Henikoff,1992, PNAS 89, 915-919). It is clear that thymine ⑴ in a DNA sequence is considered to correspond to uracil (U) in an RNA sequence when the RNA sequence is considered to be substantially similar to or have some degree of sequence identity to the DNA sequence. The percent sequence identity can be determined by sequence alignment and scoring using a computer program. Alternatively, the percent similarity or identity may also be determined by searching a database, such as FASTA, BLAST, etc.

In the present application, the term "comprises" and its variants, such as "comprising" and "comprises", generally refer to the inclusion of a stated integer or step or group of integers or steps but do not exclude the presence of any other integer or step or group of integers or steps. The term "comprising" as used herein may in turn be replaced with the term "containing" or "including," or the term "having" as used herein may sometimes be replaced with the term "having.

It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" and "an" and the terms "one or more" and "at least one" are used interchangeably herein.

Detailed Description

Isolated nucleic acid molecules

The Rep52 sequence codon is optimized, and homologous recombination of Rep78 and Rep52 is avoided after optimization. Codon optimisation may be based on codon usage of Spodoptera frugiperda organisms found in a codon usage database (see e.g.http:// www.kazusa.or.jp/codon /), and also by extracting Sf9 insect cell transcriptome sequencing data from NCBI databases, manually to extract parameters such as codon preference adaptation index. Suitable computer programs for codon optimization are available to those skilled in the art (see e.g., Anders Fuglsang,2003,Protein Expression and Purification 31：247-249;Jayaraj et al.,2005,Nucl.Acids Res.33(9):3011-3016;Chin et al.,2014 Bioinformatics 30(15)2210-2212 and on the internet). Alternatively, the optimization may be done manually using the same codon usage database. In order to avoid homologous recombination, a candidate sequence with the same number of nucleotide sequences of continuous bases being less than or equal to 30 and the homology being less than or equal to 85% is selected, and subsequent experimental verification is carried out, so that a Rep52 codon optimized sequence is finally obtained.

The nucleic acid sequence of Rep52-WT is shown as SEQ ID NO. 13, the nucleic acid sequence of Rep52-CO is shown as SEQ ID NO. 12, and the comparison between Rep52-WT and Rep52-CO codon optimization is shown in FIG. 8.

In certain embodiments, wherein the first promoter comprises a p10 promoter.

In certain embodiments, wherein the first promoter comprises a full length p10 promoter.

In certain embodiments, wherein the first promoter comprises the nucleotide sequence set forth in SEQ ID NO. 9.

In certain embodiments, wherein the second promoter comprises Polyhedrin (polh) promoter.

In certain embodiments, wherein the second promoter comprises the nucleotide sequence set forth in SEQ ID NO. 10.

In another aspect, the application provides an isolated nucleic acid molecule comprising, in order, a first polyA (pA), a nucleotide sequence encoding a Rep78 protein, a first promoter, a second promoter, a protein nucleotide sequence encoding a Rep52 protein, and a second polyA (pA), wherein the first promoter is a nucleotide sequence encoding a Rep78 protein and a transcriptional promoter of the first pA, and the second promoter is a nucleotide sequence encoding a Rep52 protein and a transcriptional promoter of the second polyA, wherein the nucleotide sequence encoding a Rep52 protein and/or the sequence of the nucleotide sequence encoding a Rep78 protein is codon optimized to avoid homologous recombination, and the first promoter has the same or higher transcriptional promoter capacity than the second promoter.

A first promoter having the same or higher transcription initiation ability as the second promoter may be defined as follows. The strength of the promoter can be determined by the expression obtained under the conditions used in the method of the application.

In certain embodiments, the first promoter or second promoter is selected from the group consisting of a polh promoter, a p10 promoter, an alkaline protein promoter, an inducible promoter or an IE1 promoter, or any other late or very late baculovirus gene promoter.

In one embodiment, the first promoter is a p10 promoter and the second promoter is a polh promoter. In another embodiment, the first promoter in the nucleic acid construct of the invention is a polh promoter and the second promoter is an IE1 promoter. In another embodiment, the first promoter in the nucleic acid construct of the invention is the pl0 promoter and the second promoter is the IE1 promoter. In another embodiment, the first promoter in the nucleic acid construct of the invention is a polh promoter and the second promoter is a polh promoter.

In certain embodiments, wherein the first promoter comprises a p10 promoter.

In certain embodiments, wherein the second promoter comprises a Polyhedrin promoter.

Vector and expression system

In certain embodiments, the vector is suitable for expression and/or replication in insect cells.

In certain embodiments, the vector comprises a viral vector.

In certain embodiments, the vector comprises a baculovirus vector, e.g., the vector may be a pFastBac vector

In certain embodiments, the vector comprises the nucleotide sequence set forth in SEQ ID NO. 14 or a nucleotide sequence having at least 50%, 60%, 70%, 80%, 81%, 82%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO. 14.

In certain embodiments, the cells comprise insect cells. For example, the cell may be an Sf9 cell.

In certain embodiments, the gene of interest comprises at least one nucleotide sequence encoding a gene product of interest expressed in a mammalian cell.

In certain embodiments, wherein the first ITR comprises at least one promoter between the first ITR and the gene of interest.

In certain embodiments, wherein the first ITR comprises at least one eukaryotic promoter between the first ITR and the gene of interest.

In certain embodiments, wherein the first ITR comprises at least one mammalian cell promoter between the first ITR and the gene of interest.

In certain embodiments, at least one nucleotide sequence encoding a gene product of interest expressed in a mammalian cell is operably linked to at least one mammalian cell compatible expression control sequence, such as a promoter. Many such promoters are known in the art. Constitutive promoters, such as the CMV promoter, which are widely expressed in a wide variety of cells, can be used. In other embodiments, the promoter is inducible, tissue specific, cell type specific, or cell cycle specific. For example, for liver-specific expression, the promoter may be selected from the group consisting of a 1-antitrypsin promoter, thyroid hormone binding globulin promoter, albumin promoter, LPS (thyroxine binding globulin) promoter, HCR-Ap0CII hybrid promoter, HCR-hAAT hybrid promoter, and apolipoprotein E promoter. Other examples include the E2F promoter for tumor-selective, in particular neural cell tumor-selective, expression (Parr et al, 1997, nat. Med. 3:1145-9) or the IL-2 promoter for use in mononuclear blood cells (Hagenbaugh et al, 1997,J Exp Med;185:2101-10).

In certain embodiments, wherein the first ITR comprises one mammalian cell promoter and one insect cell promoter between the first ITR and the gene of interest.

In certain embodiments, the promoter comprises a CMV and p10 promoter.

Insect cell

In another aspect, the application provides an insect cell comprising a first nucleotide sequence encoding a first amino acid sequence and a second nucleotide sequence encoding a second amino acid sequence, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a Rep78 protein and the second nucleotide sequence encodes a nucleotide sequence of a Rep52 protein, wherein the first nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO. 11 or a nucleotide sequence having at least 50%, 60%, 70%, 80%, 81%, 82%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO. 11, and the second nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO. 12 or a nucleotide sequence having at least 50%, 60%, 70%, 80%, 81%, 82%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO. 12.

For example, the cell line used may be from Spodoptera frugiperda (Spodoptera frugiperda), drosophila cell line, or a mosquito cell line, such as the Aedes albopictus (Aedesalbopictu) derived cell line. Preferred insect cells or cell lines are cells from insect species susceptible to baculovirus infection, including for example Se301、SeIZD2109、SeUCRU Sf9,Sf900+、Sf21、BT1-TN-5Bl-4、MG-l、Tn368、HzAml、Ha2302、Hz2E5、High Five(Invitrogen,CA,USA) and (US 6,103, 526;Protein Sciences Corp., CT, USA).

In certain embodiments, wherein the insect cell further comprises: a third nucleic acid sequence comprising at least one parvoviral Inverted Terminal Repeat (ITR) nucleotide sequence.

In the present application, the term "parvoviral ITR" is generally understood to mean a palindromic sequence comprising a majority of the sequences of the complementary, symmetrically arranged regions also known as the "C" region. The ITR functions as an origin of replication, a site that has a "cis" effect on replication, i.e., as a recognition site for a trans-acting replication protein, such as Rep78 (or Rep 68), that recognizes the palindromic structure and specific sequences within the palindromic structure. An exception to the symmetry of the ITR sequence is the "D" region of the ITR. It is unique (no complementary sequences within one ITR). Cleavage of single stranded DNA occurs at the junction between the a and D regions. It is the region where new DNA synthesis starts. The D region is typically located on one side of the palindromic structure and provides directionality to the nucleic acid replication step. Parvoviruses that replicate in mammalian cells typically contain two ITR sequences. However, it is possible to design an ITR such that the binding sites are symmetrically distributed on both strands of the A and D domains, one on each side of the palindromic structure. Thus, on a double-stranded circular DNA template (e.g., a plasmid), rep78 or Rep 68-assisted nucleic acid replication proceeds in both directions, and a single ITR sequence is sufficient for parvoviral replication of the circular vector. Thus, one ITR nucleotide sequence may be used in the context of the present application. Preferably, however, two or other even number of regular ITRs are used. Most preferably, two ITR sequences are used. In one embodiment, the parvoviral ITR is an AAV ITR.

In the present application, the term "insect cell compatible vector" is generally understood to mean a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Examples of biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any carrier may be used as long as it is compatible with insect cells. The vector may be integrated into the insect cell genome but the vector may also be episomal. The vector need not be permanently present in the insect cell, but may also include transient episomal vectors. The vector may be introduced by any known method, for example by chemical treatment, electroporation or infection of the cells. In some embodiments, the vector is a baculovirus, a viral vector, or a plasmid. For example, the vector may be a baculovirus, i.e. the construct is a baculovirus vector.

In certain embodiments, the nucleic acid construct is a baculovirus vector.

In certain embodiments, the insect cell comprises a baculovirus vector described herein.

For example, the insect cell may comprise 2 different baculoviruses: ① Bac-Rep: rep52 and Rep78 containing rAAV, respectively, regulated by the polyhedrin promoter (Polyhedrin promotor) and the p10 promoter, respectively; ② Bac-Transgene containing a Transgene surrounded by rAAV terminal repeats, regulated by a mammalian Cytomegalovirus (CMV) promoter.

For another example, the insect cell may comprise 2 different baculoviruses, each: ① Bac-Rep: rep52 and Rep78 containing rAAV, regulated by the polyhedrin promoter (Polyhedrin promotor) and the p10 promoter, respectively; ② Bac-Transgene: contains a Transgene surrounded by rAAV terminal repeats, co-regulated by insect p10 and mammalian Cytomegalovirus (CMV) promoters.

In certain embodiments, the nucleotide sequence encoding the gene of interest is positioned so that it can be integrated into neDNA that replicates in an insect cell. Any nucleotide sequence may be incorporated for subsequent expression in mammalian cells transfected with neDNA produced according to the invention. A nucleotide sequence that can express an RNAi agent, i.e., an RNA molecule capable of RNA interference, such as shRNA (short hairpin RNA) or siRNA (short interfering RNA), can be encoded, for example.

In certain embodiments, the nucleotide sequence encoding the gene of interest may encode a transposase or a defective transposon of a transposon system, including but not limited to a sleep Beauty transposon and a piggyBac transposon.

In certain embodiments, the nucleotide sequence encoding the gene of interest may encode a gene editor or a DNA template for gene editing-mediated homologous recombination, gene editing systems include, but are not limited to, CRISPR, TALEN, and various types of single base gene editors.

The product of interest expressed in mammalian cells may be a therapeutic gene product. The therapeutic gene product may be a polypeptide or RNA molecule (siRNA) or other gene product that when expressed in the target cell provides the desired therapeutic effect, e.g., eliminates unwanted activity, such as removing an infected cell or complementing a gene defect (e.g., a defect that results in a loss of enzymatic activity). Examples of therapeutic polypeptide gene products include CFTR, factor IX, factor VIII, PAH, lipoprotein lipase (LPL, preferably LPLS447X; see WO 01/00220), apolipoprotein Al, uridine diphosphate glucuronyltransferase (UGT), retinitis pigmentosa GTPase regulator interacting protein (RP-GRIP), and cytokines or interleukins such as IL-10. In certain embodiments, examples of the therapeutic gene product include a polypeptide gene therapy product encoding a therapeutic antibody. In certain embodiments, examples of the therapeutic gene products include those encoding antigens that can induce activation of humoral or cellular immune responses in vivo for the treatment of infectious diseases and tumors.

In addition, the third nucleotide sequence may also contain a nucleotide sequence encoding a polypeptide used as a marker protein to determine cell transformation and expression. Suitable marker proteins for this purpose are, for example, the fluorescent protein GFP, luciferase (Luciferase), the selectable marker gene HSV thymidine kinase (for selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection of hygromycin B), tn5 aminoglycoside phosphotransferase (for selection of G418) and dihydrofolate reductase (DHFR) (for selection of methotrexate), CD20 (low affinity nerve growth factor gene). Sources for obtaining these marker genes and methods of use thereof are described in Sambrook andRussel(2001)"Molecular Cloning:ALaboratory Manual(3rd edition),Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press,New York.

In addition, the third nucleotide sequence defined herein above may contain a nucleotide sequence encoding a polypeptide useful as a fail-safe mechanism, which polypeptide allows for the cure of a subject with cells transduced by neDNA of the present invention, if deemed necessary. Such nucleotide sequences, commonly referred to as suicide genes, encode proteins that are capable of converting the prodrug into a toxic substance that is capable of killing the transgenic cell in which the protein is expressed. Suitable examples of such suicide genes include, for example, the cytosine deaminase gene of E.coli (E.coli) or one of the thymidine kinase genes of herpes simplex virus, cytomegalovirus and varicella zoster virus, in which case ganciclovir may be used as a prodrug for killing a transgenic cell in a subject (see, for example, clair et al 1987, antimicrob. Agents chemother. 31:844-849).

In another embodiment, one gene product of interest may be an AAV protein. In particular a Rep protein, such as Rep78 and/or Rep52, or a functional fragment thereof. Expression of Rep78 and/or Rep52 in ne DNA transduced or infected mammalian cells may be beneficial for certain applications of the recombinant parvoviral (rAAV) vector by allowing long term or permanent expression of other gene products of interest introduced into the cell via the vector.

Use of the same

In certain embodiments, the method of making comprises:

2) Inserting a gene sequence of interest into said second baculovirus vector;

5) Collecting the nucleic acid molecule of interest.

In a specific operation, first, 2 kinds of baculovirus infected with Spodoptera frugiperda (Spodoptera frugiperda, sf 9) cells are respectively amplified, and the purified 2 kinds of baculovirus co-infected insect production cells are cultured in suspension. Periodically detecting the parameters of infected cell quantity, survival rate, ne DNA yield and the like in the production process so as to optimize the production process. Cells were harvested and extracted and purified neDNA for the optimal period of time, after which the yield and quality of neDNA were examined.

The application also provides the following specific embodiments:

1. An isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID No. 12.

2. The isolated nucleic acid molecule of embodiment 1, which encodes an adeno-associated virus (AAV) Rep52 protein.

3. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the adeno-associated virus (AAV) Rep78 protein and a nucleotide sequence encoding a Rep52 protein, wherein the nucleic acid molecule encoding a Rep52 protein comprises the nucleotide sequence shown in SEQ ID No. 12.

4. The isolated nucleic acid molecule of embodiment 3, wherein the nucleotide sequence encoding the Rep78 protein is wild-type.

5. The isolated nucleic acid molecule of embodiment 3, wherein the nucleic acid molecule encoding a Rep78 protein comprises the nucleotide sequence set forth in SEQ ID NO. 11.

6. The isolated nucleic acid molecule of any of embodiments 3-5, further comprising a first promoter that initiates transcription of the nucleotide sequence encoding an AAV Rep78 protein and a second promoter that initiates transcription of the nucleic acid molecule encoding an AAV Rep52 protein, the first promoter being the same as or different from the second promoter.

7. The isolated nucleic acid molecule of embodiment 6, wherein the first and second promoters comprise insect cell promoters.

8. The isolated nucleic acid molecule of any one of embodiments 6-7, wherein the first promoter comprises a strong promoter.

9. The isolated nucleic acid molecule of any one of embodiments 6-8, wherein the first promoter has the same or a higher transcription initiation capability than the second promoter.

10. The isolated nucleic acid molecule of any one of embodiments 6-9, wherein the first promoter and the second promoter are each independently selected from the group consisting of: p10 promoter, polyhedrin (polh) promoter and IE1 promoter.

11. The isolated nucleic acid molecule of any one of embodiments 6-10, wherein the transcription directions of the first and second promoters are the same or opposite.

12. The isolated nucleic acid molecule of any one of embodiments 6-11, wherein the first promoter is operably linked to the nucleotide sequence encoding the Rep78 protein and the second promoter is operably linked to the nucleotide sequence encoding the Rep52 protein.

13. The isolated nucleic acid molecule of any of embodiments 11-12, comprising, in order, a first promoter, a nucleotide sequence encoding a Rep78 protein, a second promoter, and a nucleotide sequence encoding a Rep52 protein when the transcription directions of the first promoter and the second promoter are the same.

14. The isolated nucleic acid molecule of embodiment 13, wherein the nucleotide sequence encoding the Rep78 protein and the nucleotide sequence encoding the Rep52 protein further comprise a nucleotide sequence encoding a polyA (pA), respectively.

15. The isolated nucleic acid molecule of embodiment 14, comprising, in order, a first promoter, a nucleotide sequence encoding a Rep78 protein, a first pA, a second promoter, a nucleotide sequence encoding a Rep52 protein, and a second pA when the transcription directions of the first and second promoters are the same.

16. The isolated nucleic acid molecule of any of embodiments 11-12, comprising in order a nucleotide sequence encoding Rep78, a first promoter that initiates transcription of the nucleotide sequence encoding Rep78 protein, a second promoter that initiates transcription of the nucleotide sequence encoding Rep52 protein when the transcription directions of the first and second promoters are opposite.

17. The isolated nucleic acid molecule of embodiment 16, wherein the 5 'end of the first promoter is directly or indirectly linked to the 5' end of the second promoter.

18. The isolated nucleic acid molecule of embodiment 17, wherein the 3 'end of the first promoter is directly or indirectly linked to the 5' end of the nucleotide sequence encoding Rep 78.

19. The isolated nucleic acid molecule of embodiment 18, further comprising a first pA, wherein the 3 'end of the nucleotide sequence encoding the Rep78 protein is directly or indirectly linked to the 5' end of the first pA.

20. The isolated nucleic acid molecule of embodiment 16, wherein the 3 'end of the second promoter is directly or indirectly linked to the 5' end of the nucleotide sequence encoding Rep 52.

21. The isolated nucleic acid molecule of embodiment 20, further comprising a second pA, wherein the 3 'end of the nucleotide sequence encoding Rep52 protein is directly or indirectly linked to the 5' end of the second pA.

22. The isolated nucleic acid molecule of any one of embodiments 14-21, wherein the pA is selected from the group consisting of: any one of SV40 polyA and HSV TK polyA.

23. The isolated nucleic acid molecule of any of embodiments 3-22 comprising the nucleotide sequence set forth in SEQ ID NO. 8.

24. An isolated nucleic acid molecule comprising, in order, a first polyA (pA), a nucleotide sequence encoding a Rep78 protein, a first promoter, a second promoter, a protein nucleotide sequence encoding a Rep52 protein, and a second polyA (pA), wherein the first promoter is a nucleotide sequence encoding a Rep78 protein and a transcriptional promoter of the first pA, and the second promoter is a nucleotide sequence encoding a Rep52 protein and a transcriptional promoter of the second polyA, wherein the nucleotide sequence encoding a Rep52 protein and/or the sequence of the nucleotide sequence encoding a Rep78 protein is codon optimized to avoid homologous recombination, the first promoter and the second promoter comprising insect cell promoters, the first promoter being a strong promoter.

25. The isolated nucleic acid molecule of embodiment 24, wherein the first promoter comprises a p10 promoter, a polh promoter, or an IE1 promoter.

26. The isolated nucleic acid molecule of embodiment 25, wherein the p10 promoter comprises the nucleotide sequence set forth in SEQ ID NO. 9.

27. The isolated nucleic acid molecule of embodiment 24, wherein the second promoter comprises a p10 promoter, a polh promoter, or an IE1 promoter.

28. The isolated nucleic acid molecule of embodiment 27, wherein the polh promoter comprises the nucleotide sequence set forth in SEQ ID NO. 10.

29. A vector comprising the isolated nucleic acid molecule of any one of embodiments 1-28.

30. The vector of embodiment 29, comprising a viral vector.

31. The vector of embodiment 29, comprising a baculovirus vector.

32. The vector of embodiment 29, comprising a pFastBac vector.

33. The vector according to any one of embodiments 29-32, comprising the nucleotide sequence set forth in SEQ ID NO. 14.

34. A cell comprising the isolated nucleic acid molecule of any one of embodiments 1-28 or the vector of any one of embodiments 29-33.

35. The cell of embodiment 34, comprising an insect cell.

36. The cell of embodiment 35, comprising Spodoptera frugiperda (Sf 9) cells.

37. A baculovirus expression system comprising a first baculovirus vector and a second baculovirus vector comprising a nucleic acid sequence encoding a gene of interest, said first baculovirus vector being the baculovirus vector of any one of embodiments 31-33.

38. The baculovirus expression system of claim 37, from 5 'end to 3' end, the nucleic acid sequence encoding the gene of interest comprising, in order, an inverted terminal repeat (INVERTED TERMINAL REPEAT, ITR) of the first parvovirus, the gene of interest and the second ITR.

39. The baculovirus expression system of any one of embodiment 38, wherein said first ITR further comprises at least one promoter between said first ITR and said gene of interest.

40. The baculovirus expression system of any one of embodiments 38-39, wherein said first ITR further comprises at least one eukaryotic promoter between said first ITR and said gene of interest.

41. The baculovirus expression system of any one of embodiments 38-40, wherein said first ITR further comprises at least one mammalian cell promoter between said first ITR and said gene of interest.

42. The baculovirus expression system of any one of embodiments 38-41, wherein said first ITR further comprises a mammalian cell promoter and an insect cell promoter between said first ITR and a gene of interest.

43. The baculovirus expression system of any one of embodiment 42, wherein said mammalian cell promoter comprises a broad range promoter and a tissue specific promoter.

44. The baculovirus expression system of embodiment 43, wherein said broad-spectrum promoter comprises a CMV, SV40, EF1a, CAG or UBC promoter.

45. The baculovirus expression system of embodiment 43, wherein said tissue specific promoter comprises an ALB, hAAT, TBG, TTR, GFAP, MHCK7 or hSyn promoter.

46. The baculovirus expression system of any one of embodiments 42-25, wherein said insect cell promoter comprises a p10 promoter.

47. The baculovirus expression system of any one of embodiments 42-26, said promoter comprising CMV and p10 promoters.

48. An insect cell comprising a first nucleotide sequence encoding a first amino acid sequence and a second nucleotide sequence encoding a second amino acid sequence, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a Rep78 protein and the second nucleotide sequence encodes a Rep52 protein, wherein the first nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID No. 11 and the second nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID No. 12.

49. The insect cell of embodiment 48, wherein the first and second nucleotide sequences are part of a nucleic acid construct, wherein the first and second nucleotide sequences are each operably linked to an expression control sequence for expression by the insect cell.

50. The insect cell of any one of embodiments 48-49, wherein the insect cell further comprises: a third nucleic acid sequence comprising at least one parvoviral inverted terminal repeat nucleotide sequence (INVERTED TERMINAL REPEAT, ITR).

51. The insect cell according to embodiment 50, wherein the third nucleotide sequence further comprises at least one nucleotide sequence encoding a gene of interest.

52. The insect cell of any one of embodiments 50-51, wherein the third nucleotide sequence comprises two parvoviral ITR nucleotide sequences, and wherein the at least one nucleotide sequence encoding a gene of interest is located between the two parvoviral ITR nucleotide sequences.

53. The insect cell of embodiment 52, wherein the parvovirus comprises an adeno-associated virus.

54. The insect cell according to any one of embodiments 51-53, wherein the third nucleotide sequence is part of another nucleic acid construct, wherein each nucleotide sequence encoding a gene of interest is operably linked to an expression control sequence for mammalian expression.

55. The insect cell of any one of embodiments 49-54, wherein the nucleic acid construct is an insect cell compatible vector.

56. The insect cell of embodiment 55, wherein the nucleic acid construct is a baculovirus vector.

57. The insect cell of any one of embodiments 48-56, comprising the baculovirus vector of any one of embodiments 31-33 or the baculovirus expression system of any one of embodiments 37-47.

58. Use of the baculovirus expression system of any one of embodiments 37-47 or the insect cell of any one of embodiments 48-57 in the preparation of a nucleic acid molecule of interest.

59. The use according to embodiment 58, wherein the nucleic acid molecule of interest is a linear DNA molecule (neDNA) having a covalently closed end.

60. A method of producing a nucleic acid molecule of interest comprising culturing the insect cell of any one of embodiments 48-57.

61. The method of embodiment 60, comprising:

1) Providing a baculovirus expression system of any one of embodiments 37-47;

2) Inserting a gene sequence of interest into said second baculovirus vector;

5) Collecting the nucleic acid molecule of interest.

62. The method of embodiment 62, further comprising isolating the nucleic acid molecule of interest.

63. A kit comprising the isolated nucleic acid molecule of any one of embodiments 1-28, the baculovirus expression system of any one of embodiments 37-47, and/or the insect cell of any one of embodiments 48-57.

Without intending to be limited by any theory, the following examples are presented merely to illustrate the nucleic acid molecules, vectors, expression systems, methods of preparation, uses, and the like of the present application and are not intended to limit the scope of the application.

Examples

Example 1

1. Experimental method

The technology described herein relates to the use of insect cell-baculovirus systems to prepare a linear double-stranded endless DNA (neDNA) expression vector containing AAV Inverted Terminal Repeats (ITRs) and a gene expression cassette (exemplified herein by EGFP expression cassettes). An exemplary synthetic method using neDNA vectors disclosed herein involves the following major steps:

1. Vector construction

1.1 Construction of pFastBac-ITR-EGFP donor vector

The plasmid pFastBac-ITR-EGFP was constructed by amplifying the CMV-P10-EGFP sequence by PCR using the plasmid pAAV-CMV-P10-EGFP as a template and cloning the genes into the vector pFastBac-AAV-MCS-PA by the restriction enzyme sites 5'BamHI and 3' SalI with the upstream and downstream primers P1 and P2, respectively (see Table 1).

Wherein, the pAAV-CMV-p10-EGFP vector is obtained by synthesizing CMV-p10 sequence [ SEQ ID No. 1] through genes, adding 5'KpnI and 3' NcoI enzyme cutting sites, and carrying out enzyme cutting connection and inserting into pAAV-EGFP vector [ SEQ ID No.2 ];

The pFastBac-AAV-MCS-PA vector is obtained by synthesizing an ITR-MCS-PA-ITR sequence [ SEQ ID No. 3] by genes, adding 5'KpnI and 3' HindIII cleavage sites, and inserting the cleavage ligation into the pFastBac dual vector [ SEQ ID No. 4 ].

1.2 Construction of pFastBac-ITR-Fluc donor plasmid

PFastBac-CpGfreeFluc was constructed by gene synthesis CpGfreeFluc sequence [ SEQ ID No:5], and addition of 5'SalI and 3' PmlI cleavage sites, and insertion of pFastBac-AAV-MCS-PA by cleavage ligation.

Table 1: primer sequences

Primer name	Sequence (5 '-3')	SEQ ID NO:
			P1	GATCCGGTACCACGCGTCTAG	15
P2	CTCGACGTCGACTTTACTTGTACAGC	16
			P3	GCGGGGTTTTACGAGATTGTG	17
P4	GGGGTGCCTGCTCAATCAGA	18
			P5	GCAGCACACACTGACATCCA	19
P6	GATCACCGGCGCATCAGAATTG	20
			P7	ACTTCAAGATCCGCCACAACAT	21
P8	TCTCGTTGGGGTCTTGCTCAG	22
			M13F	CCCAGTCACGACGTTGTAAAACG	23
M13R	AGCGGATAACAATTTCACACAGG	24

1.3 Construction of pFastBac-RepWT and pFastBac-inRep helper vectors

Synthesis of Gene Rep52WT [ SEQ ID No:13], plasmid pFastBac-RepWT was constructed by cloning the gene into vector pFastBac-Rep via 5'XmaI and 3' NheI. Synthesis of Gene inRep [ SEQ ID No:6], cloning of the gene into the vector pFastBac Dual by 5'BstZ17I and 3' SphI, plasmid pFastBac-inRep was constructed.

1.4 Rep52 codon optimization and construction of pFastBac-CORep helper vector

Sf9 insect cell transcriptome sequencing data were extracted from NCBI database, grasping codon bias adaptation index and codon background parameters. After the initial Rep52WT sequence is input, a child sequence is randomly generated by using a codon optimization algorithm, and iteration is circulated until the result converges, so that a candidate codon optimization gene sequence is obtained. In order to avoid homologous recombination, a candidate sequence with the same number of nucleotide sequences of continuous bases being less than or equal to 30 and the homology being less than or equal to 85% is selected, and subsequent experimental verification is carried out, so that a Rep52 codon optimized sequence is finally obtained.

Synthesis of Gene Rep52 codon optimization sequence Rep52-CO [ SEQ ID No:12], cloning of the gene into the vector pFastBac-Rep by 5'XmaI and 3' NheI, plasmid pFastBac-CORep was constructed.

1.5 Construction of pFastBac-p10Rep helper vector

The gene p10[ SEQ ID No:9] was synthesized conventionally, 5'BstZ17I and 3' NotI were added, and the gene was cloned into the vector pFastBac-CORep through 5'BstZ17I and 3' NotI to construct the plasmid pFastBac-p10Rep.

2. Plasmid transformation DH10 Bac

The donor plasmid pFastBac-ITR-EGFP or pFastBac-ITR-Fluc and helper plasmid (pFastBac-RepWT, pFastBac-inRep, pFastBac-CORep or pFastBac-p10Rep plasmid) were transformed into DH10 Bac E.coli competent cells (Solebao, cat# C1480), respectively. Recombination between the plasmid in DH10 Bac cells and the Bacmid baculovirus shuttle plasmid is induced to generate recombinant Bacmid baculovirus. The product of the Φ80dlacZ Δm15 gene in DH10 Bac cells can realize the α -complementation phenomenon of β -galactosidase for blue-white spot screening of recombinant Bacmid on LB solid medium (kanamycin (50 μg/ml), tetracycline (10 μg/ml), gentamicin (7 μg/ml), IPTG (40 μg/ml) and X-gal (100 μg/ml); white single colonies resulting from translocation of the disrupted β -galactosidase indicator gene were selected and cultured overnight at 37℃in LB medium (kanamycin (50. Mu.g/ml), tetracycline (10. Mu.g/ml) and gentamicin (7. Mu.g/ml)); recombinant Bacmid rod plasmid was extracted from E.coli using PureLink ^TM HiPure PLASIMD DNA MINIPREP KIT (Sieimer, cat# K2100-02).

3. PCR identification of recombinant Bacmid rod plasmid

The recombinant Bacmid rod plasmid was identified by PCR using the universal primer M13F/R on Bacmid (see Table 1). The conditions for PCR amplification were: 98 ℃ for 2min;98℃10s,60℃30s,72℃1min,35 cycles; and at 72℃for 5min. After the PCR is finished, agarose gel electrophoresis experiments are carried out, and the size of the target band is determined.

4. Acquisition of P0-generation recombinant baculovirus

The recombinant rod plasmids identified correctly, bacmid-ITR-EGFP, bacmid-ITR-Fluc, bacmid-RepWT, bacmid-inRep, bacmid-CORep and Bacmid-P10Rep were transfected with ExpiFectamine ^TM Sf Transfection reagent (Siemeco, cat# A38915) respectively, pre-plated Sf9 cells (Siemeco, cat# 11496-015, 27℃without CO ₂ thermostatted culture) in 6-well plates, each well containing 3ml of Sf900 ^TMIII SFM^TM medium (Siemeco, cat# 12658-019) containing 1 x 10 ⁶ Sf9 cells, continued to culture for 72-96h, when the cells appeared "vacuolated" like structure and tended to lyse, the cell culture supernatants (500 g,5 min) were collected centrifugally and passed through a 0.22 μm filter, obtaining P0-generation baculovirus, which was storable at 4℃in the dark.

Recombinant baculovirus titers were determined using the plaque method, and localized lesions formed by infected peripheral cells, depending on the replication of the virus in the infected cells. Sf9 cells were pre-plated in cell 6 well plates at 1 x 10 ⁶ cells and P0 generation baculovirus stock was serially diluted 10-fold with Sf900 ^TMIII SFM^TM medium at 10 ^-1 to 10 ^-8 dilutions, respectively, each dilution having a volume of 5ml. 1ml of each dilution was added to the above cell 6-well plate, 2 duplicate assay wells were set for each dilution, and incubated at 27℃for 1h. 10ml of 4% agar solution was prepared and thoroughly mixed with 30ml of Sf-900 medium (1.3X) (Siemens, cat# 10967-032) and placed in a 40℃water bath for use. Completely absorbing the virus diluent in the 6-hole plate, paving 2 ml/hole of the agar solution, incubating for 1h at room temperature, transferring the cell culture plate to a 27 ℃ incubator for further incubation after the agar is completely solidified. After 7-10 days, small and white spots can be seen by naked eyes, namely virus plaques, and agar in a 6-pore plate can be dyed by using 1mg/ml neutral red dye, so that the number of plaques is counted more clearly. The number of plaques individually visible at each dilution was counted and the titer of the virus was calculated using the following formula:

And simultaneously, a SYBR dye method is used for carrying out real-time quantitative PCR detection, and the copy number of the exogenous gene of the recombinant baculovirus is detected to determine the titer of the recombinant baculovirus. P0 generation baculovirus genomic DNA was extracted using GeneJET VIRAL DNA AND RNA Purification Kit (Sieimer, cat# K0821), and the viral DNA was dissolved in TE solution and stored at-80℃for use. qPCR primers were designed with EGFP, repWT, inRep, CORep and P10Rep sequences, respectively, where the RepWT, COep and P10Rep sequences shared primers P3 and P4, the inRep sequence primers were P5 and P6, and the EGFP sequence primers were P7 and P8 (see Table 1 for primer sequence information). The real-time quantitative PCR system is as follows: SYBR dye premix 25. Mu.l, upstream and downstream primers (10 μm) 2. Mu.l each, sample solution 5. Mu.l, distilled water 16. Mu.l. PCR reaction procedure: pre-denaturation 95℃60s,95℃15s,60℃15s,72℃45s,40 cycles; and (5) analyzing a dissolution curve. 3 replicate tubes were set up for each sample. According to the corresponding relation between the concentration of the standard curve and the Ct value (threshold cycle, cycle of threshold, ct), the initial concentration of each sample to be detected can be determined.

5. Amplification of recombinant baculoviruses

Typically, the P0 generation baculoviruses are small in size and low in titer, and it is necessary to continue to infect Sf9 cells to obtain high titer baculoviruses. The titer of the initial P0 generation baculovirus is 1x 10 ⁶ to 1x 10 ⁷ pfu/ml (plaque forming unit, plaque forming units, pfu), and the titer of the amplified P1 generation baculovirus is 1x 10 ⁷ to 1x 10 ⁸ pfu/ml. Using 125ml cell shake flask containing 30ml Sf9 cells (27 ℃,130 rpm), cell density being 2 x 10 ⁶ cells/ml, taking P0 generation baculovirus to infect cells according to the infection complex MOI=0.1; culturing for 72-96 hr, and centrifuging to collect cell culture supernatant (500 g,5 min) after dead cell number reaches 60-80%, and filtering with 0.22 μm filter to obtain P1 generation baculovirus. High titers of P2 virus were obtained following the same procedure as described above. Viral titers were determined using the real-time quantitative fluorescent PCR method and plaque method (supra).

6. Identification of baculovirus-expressed Bac-p10Rep protein

Western Blotting (WB) was used to detect the expression of Rep proteins. The P2-P5 generation viruses infected Sf9 cells at moi=3, respectively, and cell samples were collected by centrifugation. The cells were lysed using 1x SDS solution to prepare protein loading solutions. Electrophoresis is carried out by SDS-PAGE, and after electrophoresis is finished, protein samples are transferred to a nitrocellulose membrane; the expression of Rep protein was detected with a mouse monoclonal antibody (ARP, cat# 03-65171) to anti-AAV Rep, and the stability of the expression of Rep protein was examined, i.e., the expression of Rep protein in the P2, P3, P4 and P5-generation baculoviruses was observed.

7. Preparation of neDNA-ITR-EGFP and neDNA-ITR-Fluc expression vectors

The P2 generation two recombinant baculoviruses BacV-ITR-EGFP or BacV-ITR-Fluc and BacV-Rep are co-transfected into Sf9 insect cells of 2 x 10 ⁶ cells/ml according to MOI=1-5 (proper MOI parameters are selected), the cells are further cultured for 72-96 hours, and when the cell diameter is 18-20 mu m and the cell activity rate is about 80 percent, the cells (500 g,5 min) are collected. Small molecular weight DNA in Sf9 cells was extracted using the QIAGEN plasmid extraction kit (cat No. 12163).

8. NeDNA expression Capacity in HEK293T cells

NeDNA-ITR-EGFP was transfected with LipoFectamine 2000 transfection reagent (Siemens, cat# 11668-019) into HEK293T cells (cultured in high-sugar DMEM medium (Gibco, cat# 11965-092) containing 10% fetal bovine serum at 37℃under 5% CO ₂) and the expression of EGFP was observed using a fluorescence microscope after 72 hours.

9. Delivery of neDNA expression Capacity in C57BL/6 mice by nanolipid particles (lipid nanoparticle)

Dissolving 1mg neDNA-ITR-Fluc in sodium acetate-acetic acid buffer as an aqueous phase mixture; taking ionizable cationic lipid Dlin-MC3-DMA, dioleoyl phosphatidylcholine DOPC, cholesterol and PEG lipid to dissolve in ethanol according to the ratio of 50:10:38:2, and taking the mixture as a lipid phase mixed solution; the aqueous phase and the lipid phase are mixed by using Precision Nanosystems Ignite microfluidic chip, and then dialyzed by neutral phosphate buffer to obtain LNP-DNA complex suspension in the neutral buffer. C57BL/6 mice were injected tail vein at a dose of 2 mg/kg. When neDNA-ITR-Fluc mediated gene expression was observed, 150mg/kg of the luciferase substrate luciferin luciferin was injected intraperitoneally in mice and fluorescent signals were observed on a Xenogen IVIS Spectrum small animal biopsy imager.

2. Experimental results

1. Preparation of donor plasmid and baculovirus

An EGFP gene expression frame with ITR sequences at both ends is inserted into the plasmid pFastBac to obtain a recombinant plasmid pFastBac-ITR-EGFP (shown in FIG. 1A).

Construction of helper plasmid pFastBac-p10Rep (FIG. 1B, SEQ ID NO: 14), p10 being the promoter of Rep78-WT and polh being the promoter of Rep 52-CO. Helper plasmid pFastBac-RepWT was constructed, where ΔIE1 is the promoter of Rep78 and polh is the promoter of Rep52 WT. Helper plasmid pFastBac-inRep was constructed, in which p10 was the Rep78 promoter, with a synthetic Intron (Intron) sequence (containing polh promoter) between Rep78 and Rep52, in the same expression cassette, as promoter for Rep52, based on HAIFENG CHEN's 2008 work. Helper plasmid pFastBac-CORep was constructed, ΔIE1 was the promoter for Rep78 and polh was the promoter for Rep 52-CO. FIG. 2 is a schematic diagram of transcription of the p10Rep gene, repWT gene, inRep gene and CORep gene.

The donor plasmid (pFastBac-ITR-EGFP) and different auxiliary plasmids are respectively transformed into DH10 Bac escherichia coli competent cells, and the Bacmid-ITR-EGFP and Bacmid-Rep are obtained by screening blue spots, and recombinant Bacmid rod plasmids with correct sequences are further screened by PCR. And (3) respectively transfecting Sf9 cells by the recombinant Bacmid to obtain recombinant baculoviruses BacV-ITR-EGFP and BacV-Rep.

2. Expression of Rep proteins in insect cells Sf9

The Rep protein expressed by BacV-p10 Rep-infected Sf9 cells was detected with anti-AAV Rep mouse monoclonal antibody (ARP, cat# 03-65171), while uninfected Sf9 cells were used as negative controls. BacV-RepWT ^[1],BacV-inRep^[2] and BacV-CORep (Rep 52 sequence in optimized RepWT) were used as controls. The P1 generation BacV-Rep baculovirus was serially infected with Sf9 cells at moi=0.1 to obtain P2, P3, P4 and P5 baculovirus, respectively. Sf9 cells were infected with P2-P5 generation BacV-Rep at moi=3, cell samples were collected, expression of Rep proteins was detected with WB, and stability of Rep protein expression in different generation baculoviruses was examined as shown in fig. 3.

3. Preparation of neDNA-ITR-EGFP Gene expression vector

The P2 generation two recombinant baculoviruses BacV-ITR-EGFP and BacV-Rep are used for co-transfecting Sf9 insect cells according to MOI=3, culturing for 72-96h, collecting the cells when the cell diameter is 18-20 mu m and the cell activity rate is about 80%, and extracting neDNA-ITR-EGFP gene expression vectors with small molecular weight. The band size of neDNA was identified by electrophoresis on a 0.8% agarose gel, with major bands at 2.7kb and 5.4kb, corresponding to the monomers and dimers of the neDNA-ITR-EGFP expression vector, respectively. The bands above the dimer can be extrapolated to trimers and multimers depending on size, as shown in FIG. 4.

NeDNA was digested with SalI single restriction enzyme to give a 2kb,0.7kb band, which was consistent with the expected fragment size after cleavage of the monomer, as shown in FIGS. 5A-B, the 5A.neDNA-EGFP monomer fragment was 2.7kb long, and SalI restriction enzyme was able to cleave the monomer fragment into fragments of 2kb and 0.7kb in size. The dimer has a structure of "head-to-head, tail-to-tail", and can be cleaved into fragments of 2kb and 1.4kb in size or fragments of 4kb and 0.7kb in size. Dark blue rectangles indicate 5'itr sequences and light blue rectangles 3' itr sequences.

The expression yields of the different Rep protein driven neDNA-ITR-EGFP gene expression vectors were different, and the expression yields of the p10Rep driven neDNA-ITR-EGFP gene expression vectors were about: approximately 270 μg of neDNA gene expression vector was expressed in each 6×10 ⁷ Sf9 cells, 2-3 fold compared to the other three groups, as detailed in table 2.

Table 2: neDNA yield

4. NeDNA-ITR-EGFP gene expression vector transfected HEK293 cells

NeDNA-ITR-EGFP was transfected into HEK293 cells with LipoFectamine 2000 transfection reagent and expression of EGFP was observed using a fluorescence microscope beginning 24 h. 72h after transfection, the expression of the nedna-ITR-EGFP gene expression vector in HEK293 cells is shown in fig. 6.

5. NeDNA-ITR-Fluc gene expression vector for expressing luciferase in mice

The baculovirus BacV-ITR-Fluc and BacV-p10Rep were obtained by constructing Bacmid-ITR-Fluc and Bacmid-p10Rep using the donor plasmid pFastBac-ITR-Fluc and the helper plasmid pFastBac-p10Rep, and neDNA-ITR-Fluc gene expression vectors were prepared. Delivery with nanolipid particles (LNP) neDNA-ITR-Fluc tail intravenous injection in C57BL/6 mice stable expression of luciferase in the mouse liver was observed at 24h start, as shown in FIG. 7.

In conclusion, the method comprises the steps of,

The present application provides a method for producing neDNA using an insect cell-baculovirus expression system, using which neDNA is produced in a variety of configurations, such as: monomers, dimers, trimers, and multimers, and the like. In mouse experiments, the neDNA may mediate long-lasting expression of the gene expression cassette of interest in vivo (e.g., liver) as compared to plasmid DNA.

The method optimizes the Rep protein expression vector, improves the expression stability of the Rep protein, thereby improving the yield of neDNA and the stability of a production system, wherein p10Rep is an optimized expression frame, a complete p10 promoter and the expression quantity of Rep 78; after Rep52 sequence codon optimization, homologous recombination between Rep78 and Rep52 is avoided.

Compared with other Bac-Rep and Bac-EFGP co-infected Sf9 cells, the Bac-p10Rep has the highest neDNA yield, and the average yield is improved by 2-3 times.

Compared with other Bac-Rep, the expression stability of the Rep protein (Rep 78) is better after 3 continuous baculovirus passages.

Reference is made to:

[1]Masashi Urabe,Chuantian Ding,Robert M Kotin.Insect cells as a factory to produce adeno-associated virus type 2 vectors.Hum Gene Ther,2002,13(16):1935-43.

[2]Haifeng Chen.Intron splicing-mediated expression of AAV Rep and Cap genes and production of AAV vectors in insect cells.Mol Ther,2008,16(5):924-30.

sequence listing

<110> Bohai due to Biotechnology Co., ltd

<120> Baculovirus vector and use thereof

<130> 0251-PA-002

<160> 24

<170> PatentIn version 3.5

<210> 1

<211> 1097

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> CMV-p10

<400> 1

ggtaccacgc gtctagttat taatagtaat caattacggg gtcattagtt catagcccat 60

atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 120

acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 180

tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 240

tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 300

attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc tacgtattag 360

tcatcgctat taccatgctg atgcggtttt ggcagtacac caatgggcgt ggatagcggt 420

ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 480

accaaaatca acgggacttt ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg 540

gcggtaggcg tgtacggtgg gaggtctata taagcagacg tcgtttagtg aaccgtcaga 600

tcactagatg ctttattgcg gtagtttatc acagttaaat tgctaacgcc agtctcgaac 660

ttaacgtgca gaagttggtc gtgaggcact gggcaggtaa gtatcgggcc ctttgtgcgg 720

ggggagcggc tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg 780

cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg 840

ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt 900

ttggcaaaga attggatcgg accgaaatta atacgactca ctatagggga attgtgagcg 960

gataacaatt ccccggagtt aatccgggac ctttaattca acccaacaca atatattata 1020

gttaaataag aattattatc aaatcatttg tatattaatt aaaatactat actgtaaatt 1080

acattttatt tacaatc 1097

<210> 2

<211> 5547

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> pAAV-GFP

<400> 2

cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60

cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120

gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180

agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240

acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300

acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360

agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420

gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480

cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540

cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600

tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660

gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720

tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780

ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840

taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900

cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960

acgattaccg ttcatcgcct gcactgcgcg ctcgctcgct cactgaggcc gcccgggcaa 1020

agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag 1080

agggagtgga attcacgcgt ggtacgatct gaattcggta caattcacgc gtgggtacca 1140

cgcgtctagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga 1200

gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 1260

cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 1320

acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 1380

tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 1440

ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 1500

tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc 1560

acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa 1620

tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag 1680

gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg 1740

gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctcca 1800

ccggttcgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 1860

ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg 1920

cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 1980

gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc 2040

cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 2100

gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 2160

gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 2220

catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga 2280

caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag 2340

cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 2400

gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg 2460

cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 2520

gctgtacaag taaagcggcc atcaagctta tcgataccgt cgactagagc tcgctgatca 2580

gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 2640

ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 2700

cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 2760

gaggattggg aagacaatag caggcatgct ggggagagat cgatctgagg aacccctagt 2820

gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa 2880

ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagaga 2940

gggagtggcc aacccccccc cccccccccc tgcatgcagg cgattctctt gtttgctcca 3000

gactctcagg caatgacctg atagcctttg tagagacctc tcaaaaatag ctaccctctc 3060

cggcatgaat ttatcagcta gaacggttga atatcatatt gatggtgatt tgactgtctc 3120

cggcctttct cacccgtttg aatctttacc tacacattac tcaggcattg catttaaaat 3180

atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc ccgcaaaagt 3240

attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg aggctttatt 3300

gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgttg gaattcctga 3360

tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca 3420

gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg 3480

acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 3540

ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg 3600

gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt 3660

caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 3720

attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 3780

aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 3840

tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 3900

agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 3960

gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 4020

cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactgagtga 4080

taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 4140

tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 4200

agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 4260

caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 4320

ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 4380

tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 4440

agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 4500

tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 4560

agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 4620

gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 4680

gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 4740

tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 4800

gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 4860

accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 4920

accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 4980

gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 5040

ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 5100

atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 5160

gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 5220

cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 5280

gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 5340

gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 5400

tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 5460

cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 5520

ccccgcgcgt tggccgattc attaatg 5547

<210> 3

<211> 925

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> ITR-MCS-PA-ITR

<400> 3

ggtaccacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa 60

agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag 120

agggagtggc caactccatc actaggggtt cctgcggccg cagatctacc ggtggcgcgc 180

cggatccgaa ttctctagag tcgacgtcga gctagcatcg atgtttaaac gagctcacta 240

gtctcgagac gcgtacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct 300

ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt 360

gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg 420

gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc 480

agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc 540

tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt 600

tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca 660

ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc 720

cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc 780

ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 840

ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 900

agctgcctgc aggggcgcca agctt 925

<210> 4

<211> 5238

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> pFastBac dual

<400> 4

ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60

ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 420

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 480

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 540

attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 600

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 660

gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 720

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 780

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 840

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 900

agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 960

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 1020

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 1080

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 1140

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 1200

accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 1260

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 1320

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 1380

taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 1440

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1500

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1560

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1620

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1680

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1740

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1800

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1860

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1920

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1980

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 2040

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 2100

acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 2160

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 2220

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 2280

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 2340

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 2400

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 2460

cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2520

tctgtgcggt atttcacacc gcagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2580

agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2640

caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2700

aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2760

cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2820

gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2880

gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2940

ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 3000

ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3060

cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3120

ctcactacgc ggctgctcaa acctgggcag aacgtaagcc gcgagagcgc caacaaccgc 3180

ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3240

atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3300

atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3360

gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3420

gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg 3480

cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3540

aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3600

agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc 3660

gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3720

aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780

cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg ccctggcttc 3840

aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3900

tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3960

atagttctag tggttggcct acgtacccgt agtggctatg gcagggcttg ccgccccgac 4020

gttggctgcg agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga 4080

aacgcgggcg tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg 4140

ggaccgaacc ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt 4200

ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc 4260

tcccccatct cccggtaccg catgctatgc atcagctgct agcaccatgg ctcgagatcc 4320

cgggtgatca agtcttcgtc gagtgattgt aaataaaatg taatttacag tatagtattt 4380

taattaatat acaaatgatt tgataataat tcttatttaa ctataatata ttgtgttggg 4440

ttgaattaaa ggtccgtata ctccggaata ttaatagatc atggagataa ttaaaatgat 4500

aaccatctcg caaataaata agtattttac tgttttcgta acagttttgt aataaaaaaa 4560

cctataaata ttccggatta ttcataccgt cccaccatcg ggcgcggatc ccggtccgaa 4620

gcgcgcggaa ttcaaaggcc tacgtcgacg agctcactag tcgcggccgc tttcgaatct 4680

agagcctgca gtctcgacaa gcttgtcgag aagtactaga ggatcataat cagccatacc 4740

acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa 4800

cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa 4860

taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 4920

ggtttgtcca aactcatcaa tgtatcttat catgtctgga tctgatcact gcttgagcct 4980

aggagatccg aaccagataa gtgaaatcta gttccaaact attttgtcat ttttaatttt 5040

cgtattagct tacgacgcta cacccagttc ccatctattt tgtcactctt ccctaaataa 5100

tccttaaaaa ctccatttcc acccctccca gttcccaact attttgtccg cccacagcgg 5160

ggcatttttc ttcctgttat gtttttaatc aaacatcctg ccaactccat gtgacaaacc 5220

gtcatcttcg gctacttt 5238

<210> 5

<211> 3129

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> CpGfreeFluc

<400> 5

tttagggtta gggttagggt tagggaaaaa tttagggtta gggttagggt tagggaaaaa 60

tttagggtta gggttagggt tagggaaaaa aagcttgagt caatgggaaa aacccattgg 120

agccaagtac actgactcaa tagggacttt ccattgggtt ttgcccagta cataaggtca 180

atagggggtg agtcaacagg aaagtcccat tggagccaag tacattgagt caatagggac 240

tttccaatgg gttttgccca gtacataagg tcaatgggag gtaagccaat gggtttttcc 300

cattactgac atgtatactg agtcattagg gactttccaa tgggttttgc ccagtacata 360

aggtcaatag gggtgaatca acaggaaagt cccattggag ccaagtacac tgagtcaata 420

gggactttcc attgggtttt gcccagtaca aaaggtcaat agggggtgag tcaatgggtt 480

tttcccatta ttggcacata cataaggtca ataggggtga ctagtggaga agagcatgct 540

tgagggctga gtgcccctca gtgggcagag agcacatggc ccacagtccc tgagaagttg 600

gggggagggg tgggcaattg aactggtgcc tagagaaggt ggggcttggg taaactggga 660

aagtgatgtg gtgtactggc tccacctttt tccccagggt gggggagaac catatataag 720

tgcagtagtc tctgtgaaca ttcaagcttc tgccttctcc ctcctgtgag tttggtaagt 780

cactgactgt ctatgcctgg gaaagggtgg gcaggaggtg gggcagtgca ggaaaagtgg 840

cactgtgaac cctgcagccc tagacaattg tactaacctt cttctctttc ctctcctgac 900

aggttggtgt acagtagctt ccaccatgga ggatgccaag aatattaaga aaggccctgc 960

cccattctac cctctggaag atggcactgc tggtgagcaa ctgcacaagg ccatgaagag 1020

gtatgccctg gtccctggca ccattgcctt cactgatgct cacattgagg tggacatcac 1080

ctatgctgaa tactttgaga tgtctgtgag gctggcagaa gccatgaaaa gatatggact 1140

gaacaccaac cacaggattg tggtgtgctc tgagaactct ctccagttct tcatgcctgt 1200

gttaggagcc ctgttcattg gagtggctgt ggcccctgcc aatgacatct acaatgagag 1260

agagctcctg aacagcatgg gcatcagcca gccaactgtg gtctttgtga gcaagaaggg 1320

cctgcaaaag atcctgaatg tgcagaagaa gctgcccatc atccagaaga tcatcatcat 1380

ggacagcaag actgactacc agggcttcca gagcatgtat acctttgtga ccagccactt 1440

accccctggc ttcaatgagt atgactttgt gcctgagagc tttgacaggg acaagaccat 1500

tgctctgatt atgaacagct ctggctccac tggactgccc aaaggtgtgg ctctgcccca 1560

cagaactgct tgtgtgagat tcagccatgc cagagacccc atctttggca accagatcat 1620

ccctgacact gccatcctgt ctgtggttcc attccatcat ggctttggca tgttcacaac 1680

actggggtac ctgatctgtg gcttcagagt ggtgctgatg tataggtttg aggaggagct 1740

gtttctgagg agcctacaag actacaagat ccagtctgcc ctgctggtgc ccactctgtt 1800

cagcttcttt gccaagagca ccctcattga caagtatgac ctgagcaacc tgcatgagat 1860

tgcctctgga ggagcacccc tgagcaagga ggtgggtgag gctgtggcaa agaggttcca 1920

tctcccagga atcagacagg gctatggcct gactgagacc acctctgcca tcctcatcac 1980

ccctgaagga gatgacaagc ctggtgctgt gggcaaggtg gttccctttt ttgaggccaa 2040

ggtggtggac ctggacactg gcaagaccct gggagtgaac cagaggggtg agctgtgtgt 2100

gaggggtccc atgatcatgt ctggctatgt gaacaaccct gaggccacca atgccctgat 2160

tgacaaggat ggctggctgc actctggtga cattgcctac tgggatgagg atgagcactt 2220

tttcattgtg gacaggctga agagcctcat caagtacaaa ggctaccaag tggcacctgc 2280

tgagctagag agcatcctgc tccagcaccc caacatcttt gatgctggtg tggctggcct 2340

gcctgatgat gatgctggag agctgcctgc tgctgttgtg gttctggagc atggaaagac 2400

catgactgag aaggagattg tggactatgt ggccagtcag gtgaccactg ccaagaagct 2460

gaggggaggt gtggtgtttg tggatgaggt gccaaagggt ctgactggca agctggatgc 2520

cagaaagatc agagagatcc tgatcaaggc caagaagggt ggcaaacaat tgatctctgg 2580

agccaatgga gtctagctag ctggccagac atgataagat acattgatga gtttggacaa 2640

accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2700

ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2760

atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa 2820

tgtggtatgg aattcggatc cggtgtggaa agtccccagg ctccccagca ggcagaagta 2880

tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag 2940

caggcagaag tatgcaaagc atgcatctca attagtcagc aaccagagct ctggggactt 3000

tccgctgggg actttccgct ggggactttc cgctggggac tttccgctgg ggactttccg 3060

catttaaatg gtacattttg ttctagaaca aaatgtaccg gtacattttg ttctggtaca 3120

ttttgttct 3129

<210> 6

<211> 2305

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> inRep

<400> 6

gacctttaat tcaacccaac acaatatatt atagttaaat aagaattatt atcaaatcat 60

ttgtatatta attaaaatac tatactgtaa attacatttt atttacaatc actcgacgaa 120

gacttgatca ccctacccgc catgccgggg ttttacgaga ttgtgattaa ggtccccagc 180

gaccttgacg agcatctgcc cggcatttct gacagctttg tgaactgggt ggccgagaag 240

gaatgggagt tgccgccaga ttctgacatg gatctgaatc tgattgagca ggcacccctg 300

accgtggccg agaagctgca gcgcgacttt ctgacggaat ggcgccgtgt gagtaaggcc 360

ccggaggccc ttttctttgt gcaatttgag aagggagaga gctacttcca catgcacgtg 420

ctcgtggaaa ccaccggggt gaaatccatg gttttgggac gtttcctgag tcagattcgc 480

gaaaaactga ttcagagaat ttaccgcggg atcgagccga ctttgccaaa ctggttcgcg 540

gtcacaaaga ccagaaatgg cgccggaggc gggaacaagg tggtggatga gtgctacatc 600

cccaattact tgctccccaa aacccagcct gagctccagt gggcgtggac taatatggaa 660

cagtatttaa ggtaagtact ccctatcagt gatagagatc tatcatggag ataattaaaa 720

tgataaccat ctcgcaaata aataagtatt ttactgtttt cgtaacagtt ttgtaataaa 780

aaaacctata aatattccgg attattcata ccgtcccacc atcgggcgcg aagggggaga 840

cctgtagtca gagcccccgg gcagcacaca ctgacatcca ctcccttcct attgtttcag 900

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc 960

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag 1020

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac 1080

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc 1140

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac 1200

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg 1260

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct 1320

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac 1380

taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt 1440

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg 1500

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag 1560

caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat 1620

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca 1680

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga 1740

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt 1800

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc 1860

cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac 1920

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca 1980

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc 2040

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc 2100

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat 2160

gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg 2220

catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt 2280

ggctcgagga cactctctct gaagg 2305

<210> 7

<211> 3884

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> CORep

<400> 7

gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg 60

gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct cccggtaccg 120

catgctatgc atcagctgct agcttactgc tcgaagatgc agtcgtccag atccacgttc 180

acgagatcgc aagcagtgca agcgtcgggc accttgccca tgatgtggtg gatgtagcac 240

agcttctggt aggccttctt gacgacggac acgggttggg actcggagac ggggaagcat 300

tccagacagt ctttctggcc gtgggtgaag cagatgttgg agttctggtt catgcgctcg 360

cactggcggc aagggaacag catcagattc atgcccacgt ggcggctgca cttattctgg 420

tagcggtcgg cgtagttgat ggaggcttca gcatcggagg tggaaggctg agcgacggac 480

tcacgcacgc gcttaggctc gctgatatca gcgtcgctgg gagcggggcg cttcttagca 540

ccgcccttct tcacgtagaa ctcgtgctcc acctccacga cgtgatcctt ggcccaacgg 600

aagaagtcct tcacctcttg cttggtgact ttgccgaagt cgtggtccag acggcgggtg 660

agctcgaatt tgaacatgcg gtcttgcaga ggttgctgat gttcgaaggt agtggagttg 720

ccgtcgatga cagcgcacat gttggtgttg gaagtcacga tgacgggggt ggggtcgatc 780

tgagcggagg acttgcactt ctggtcgaca cgcaccttgc taccacccag aatggccttg 840

gcggattcga ccaccttggc agtcatcttg ccctcttccc accagatgac catcttgtcg 900

acgcagtcgt tgaaggggaa gttctcgttg gtccagttga cgcagccgta aaagggcacg 960

gtatgggcga tggcttcggc gatgttggtc ttaccagtgg tagcgggacc gaagagccag 1020

atagtgttgc gcttgccgaa cttcttggta gcccaaccga ggaagacgga ggcggcatac 1080

tgggggtcgt agccgttgag ctccagaatc ttgtagatgc ggttggagga gatgtcctcc 1140

acgggctgtt gaccgaccag ataatcggga gcggtcttgg tgaggctcat gatcttacca 1200

gcgttgtcga gggcagcctt gatctgggaa cgggagttgc tggcagcatt gaagctgatg 1260

tagctggctt ggtcctcttg gatccactgc ttctcgctag tgatgccctt gtcgaccagc 1320

caaccgacca gttccatggt ggcccgggtt tcggaccgag atccgcgccc gatggtggga 1380

cggtatgaat aatccggaat atttataggt ttttttatta caaaactgtt acgaaaacag 1440

taaaatactt atttatttgc gagatggtta tcattttaat tatctccatg atctattaat 1500

attccggagt atacaataaa cgataacgcc gttggtggcg tgaggcatgt aaaaggttac 1560

atcattatct tgttcgccat ccggttggta taaatagacg ttcatgttgg tttttgtttc 1620

agttgcaagt tggctgcggc gcgcgcagca cctttgcggc cgccaccatg gcggggtttt 1680

acgagattgt gattaaggtc cccagcgacc ttgacgagca tctgcccggc atttctgaca 1740

gctttgtgaa ctgggtggcc gagaaggaat gggagttgcc gccagattct gacatggatc 1800

tgaatctgat tgagcaggca cccctgaccg tggccgagaa gctgcagcgc gactttctga 1860

cggaatggcg ccgtgtgagt aaggccccgg aggccctttt ctttgtgcaa tttgagaagg 1920

gagagagcta cttccacatg cacgtgctcg tggaaaccac cggggtgaaa tccatggttt 1980

tgggacgttt cctgagtcag attcgcgaaa aactgattca gagaatttac cgcgggatcg 2040

agccgacttt gccaaactgg ttcgcggtca caaagaccag aaatggcgcc ggaggcggga 2100

acaaggtggt ggatgagtgc tacatcccca attacttgct ccccaaaacc cagcctgagc 2160

tccagtgggc gtggactaat atggaacagt atttaagcgc ctgtttgaat ctcacggagc 2220

gtaaacggtt ggtggcgcag catctgacgc acgtgtcgca gacgcaggag cagaacaaag 2280

agaatcagaa tcccaattct gatgcgccgg tgatcagatc aaaaacttca gccaggtaca 2340

tggagctggt cgggtggctc gtggacaagg ggattacctc ggagaagcag tggatccagg 2400

aggaccaggc ctcatacatc tccttcaatg cggcctccaa ctcgcggtcc caaatcaagg 2460

ctgccttgga caatgcggga aagattatga gcctgactaa aaccgccccc gactacctgg 2520

tgggccagca gcccgtggag gacatttcca gcaatcggat ttataaaatt ttggaactaa 2580

acgggtacga tccccaatat gcggcttccg tctttctggg atgggccacg aaaaagttcg 2640

gcaagaggaa caccatctgg ctgtttgggc ctgcaactac cgggaagacc aacatcgcgg 2700

aggccatagc ccacactgtg cccttctacg ggtgcgtaaa ctggaccaat gagaactttc 2760

ccttcaacga ctgtgtcgac aagatggtga tctggtggga ggaggggaag atgaccgcca 2820

aggtcgtgga gtcggccaaa gccattctcg gaggaagcaa ggtgcgcgtg gaccagaaat 2880

gcaagtcctc ggcccagata gacccgactc ccgtgatcgt cacctccaac accaacatgt 2940

gcgccgtgat tgacgggaac tcaacgacct tcgaacacca gcagccgttg caagaccgga 3000

tgttcaaatt tgaactcacc cgccgtctgg atcatgactt tgggaaggtc accaagcagg 3060

aagtcaaaga ctttttccgg tgggcaaagg atcacgtggt tgaggtggag catgaattct 3120

acgtcaaaaa gggtggagcc aagaaaagac ccgcccccag tgacgcagat ataagtgagc 3180

ccaaacgggt gcgcgagtca gttgcgcagc catcgacgtc agacgcggaa gcttcgatca 3240

actacgcaga caggtaccaa aacaaatgtt ctcgtcacgt gggcatgaat ctgatgctgt 3300

ttccctgcag acaatgcgag agaatgaatc agaattcaaa tatctgcttc actcacggac 3360

agaaagactg tttagagtgc tttcccgtgt cagaatctca acccgtttct gtcgtcaaaa 3420

aggcgtatca gaaactgtgc tacattcatc atatcatggg aaaggtgcca gacgcttgca 3480

ctgcctgcga tctggtcaat gtggatttgg atgactgcat ctttgaacaa taaatgattt 3540

aaatcaggta tggctgccga tggttatctt ccagattggc tcgaggacac tctctctgat 3600

ctagagcctg cagtctcgac aagcttgtcg agaagtacta gaggatcata atcagccata 3660

ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 3720

aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca 3780

aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 3840

gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatc 3884

<210> 8

<211> 3874

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> p10Rep

<400> 8

gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg 60

gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct cccggtaccg 120

catgctatgc atcagctgct agcttactgc tcgaagatgc agtcgtccag atccacgttc 180

acgagatcgc aagcagtgca agcgtcgggc accttgccca tgatgtggtg gatgtagcac 240

agcttctggt aggccttctt gacgacggac acgggttggg actcggagac ggggaagcat 300

tccagacagt ctttctggcc gtgggtgaag cagatgttgg agttctggtt catgcgctcg 360

cactggcggc aagggaacag catcagattc atgcccacgt ggcggctgca cttattctgg 420

tagcggtcgg cgtagttgat ggaggcttca gcatcggagg tggaaggctg agcgacggac 480

tcacgcacgc gcttaggctc gctgatatca gcgtcgctgg gagcggggcg cttcttagca 540

ccgcccttct tcacgtagaa ctcgtgctcc acctccacga cgtgatcctt ggcccaacgg 600

aagaagtcct tcacctcttg cttggtgact ttgccgaagt cgtggtccag acggcgggtg 660

agctcgaatt tgaacatgcg gtcttgcaga ggttgctgat gttcgaaggt agtggagttg 720

ccgtcgatga cagcgcacat gttggtgttg gaagtcacga tgacgggggt ggggtcgatc 780

tgagcggagg acttgcactt ctggtcgaca cgcaccttgc taccacccag aatggccttg 840

gcggattcga ccaccttggc agtcatcttg ccctcttccc accagatgac catcttgtcg 900

acgcagtcgt tgaaggggaa gttctcgttg gtccagttga cgcagccgta aaagggcacg 960

gtatgggcga tggcttcggc gatgttggtc ttaccagtgg tagcgggacc gaagagccag 1020

atagtgttgc gcttgccgaa cttcttggta gcccaaccga ggaagacgga ggcggcatac 1080

tgggggtcgt agccgttgag ctccagaatc ttgtagatgc ggttggagga gatgtcctcc 1140

acgggctgtt gaccgaccag ataatcggga gcggtcttgg tgaggctcat gatcttacca 1200

gcgttgtcga gggcagcctt gatctgggaa cgggagttgc tggcagcatt gaagctgatg 1260

tagctggctt ggtcctcttg gatccactgc ttctcgctag tgatgccctt gtcgaccagc 1320

caaccgacca gttccatggt ggcccgggtt tcggaccgag atccgcgccc gatggtggga 1380

cggtatgaat aatccggaat atttataggt ttttttatta caaaactgtt acgaaaacag 1440

taaaatactt atttatttgc gagatggtta tcattttaat tatctccatg atctattaat 1500

attccggagt atacggacct ttaattcaac ccaacacaat atattatagt taaataagaa 1560

ttattatcaa atcatttgta tattaattaa aatactatac tgtaaattac attttattta 1620

caatcactcg acgaagactt gatcagcggc cgccaccatg gcggggtttt acgagattgt 1680

gattaaggtc cccagcgacc ttgacgagca tctgcccggc atttctgaca gctttgtgaa 1740

ctgggtggcc gagaaggaat gggagttgcc gccagattct gacatggatc tgaatctgat 1800

tgagcaggca cccctgaccg tggccgagaa gctgcagcgc gactttctga cggaatggcg 1860

ccgtgtgagt aaggccccgg aggccctttt ctttgtgcaa tttgagaagg gagagagcta 1920

cttccacatg cacgtgctcg tggaaaccac cggggtgaaa tccatggttt tgggacgttt 1980

cctgagtcag attcgcgaaa aactgattca gagaatttac cgcgggatcg agccgacttt 2040

gccaaactgg ttcgcggtca caaagaccag aaatggcgcc ggaggcggga acaaggtggt 2100

ggatgagtgc tacatcccca attacttgct ccccaaaacc cagcctgagc tccagtgggc 2160

gtggactaat atggaacagt atttaagcgc ctgtttgaat ctcacggagc gtaaacggtt 2220

ggtggcgcag catctgacgc acgtgtcgca gacgcaggag cagaacaaag agaatcagaa 2280

tcccaattct gatgcgccgg tgatcagatc aaaaacttca gccaggtaca tggagctggt 2340

cgggtggctc gtggacaagg ggattacctc ggagaagcag tggatccagg aggaccaggc 2400

ctcatacatc tccttcaatg cggcctccaa ctcgcggtcc caaatcaagg ctgccttgga 2460

caatgcggga aagattatga gcctgactaa aaccgccccc gactacctgg tgggccagca 2520

gcccgtggag gacatttcca gcaatcggat ttataaaatt ttggaactaa acgggtacga 2580

tccccaatat gcggcttccg tctttctggg atgggccacg aaaaagttcg gcaagaggaa 2640

caccatctgg ctgtttgggc ctgcaactac cgggaagacc aacatcgcgg aggccatagc 2700

ccacactgtg cccttctacg ggtgcgtaaa ctggaccaat gagaactttc ccttcaacga 2760

ctgtgtcgac aagatggtga tctggtggga ggaggggaag atgaccgcca aggtcgtgga 2820

gtcggccaaa gccattctcg gaggaagcaa ggtgcgcgtg gaccagaaat gcaagtcctc 2880

ggcccagata gacccgactc ccgtgatcgt cacctccaac accaacatgt gcgccgtgat 2940

tgacgggaac tcaacgacct tcgaacacca gcagccgttg caagaccgga tgttcaaatt 3000

tgaactcacc cgccgtctgg atcatgactt tgggaaggtc accaagcagg aagtcaaaga 3060

ctttttccgg tgggcaaagg atcacgtggt tgaggtggag catgaattct acgtcaaaaa 3120

gggtggagcc aagaaaagac ccgcccccag tgacgcagat ataagtgagc ccaaacgggt 3180

gcgcgagtca gttgcgcagc catcgacgtc agacgcggaa gcttcgatca actacgcaga 3240

caggtaccaa aacaaatgtt ctcgtcacgt gggcatgaat ctgatgctgt ttccctgcag 3300

acaatgcgag agaatgaatc agaattcaaa tatctgcttc actcacggac agaaagactg 3360

tttagagtgc tttcccgtgt cagaatctca acccgtttct gtcgtcaaaa aggcgtatca 3420

gaaactgtgc tacattcatc atatcatggg aaaggtgcca gacgcttgca ctgcctgcga 3480

tctggtcaat gtggatttgg atgactgcat ctttgaacaa taaatgattt aaatcaggta 3540

tggctgccga tggttatctt ccagattggc tcgaggacac tctctctgat ctagagcctg 3600

cagtctcgac aagcttgtcg agaagtacta gaggatcata atcagccata ccacatttgt 3660

agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat 3720

gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa 3780

tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 3840

caaactcatc aatgtatctt atcatgtctg gatc 3874

<210> 9

<211> 110

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> p10

<400> 9

gacctttaat tcaacccaac acaatatatt atagttaaat aagaattatt atcaaatcat 60

ttgtatatta attaaaatac tatactgtaa attacatttt atttacaatc 110

<210> 10

<211> 92

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> polh

<400> 10

atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc 60

gtaacagttt tgtaataaaa aaacctataa at 92

<210> 11

<211> 1866

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> Rep78-WT

<400> 11

atggcggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc 60

ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat 120

tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 180

cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg 240

caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg 300

aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt 360

taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaatggc 420

gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctccccaaa 480

acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag cgcctgtttg 540

aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 600

gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag atcaaaaact 660

tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag 720

cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg 780

tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc 840

cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg gatttataaa 900

attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc 960

acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag 1020

accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc 1080

aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg 1140

aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc 1200

gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc 1260

aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg 1320

ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag 1380

gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg 1440

gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca 1500

gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg 1560

gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg 1620

aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc 1680

ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt 1740

tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg 1800

ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa 1860

caataa 1866

<210> 12

<211> 1194

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> Rep52-CO

<400> 12

atggaactgg tcggttggct ggtcgacaag ggcatcacta gcgagaagca gtggatccaa 60

gaggaccaag ccagctacat cagcttcaat gctgccagca actcccgttc ccagatcaag 120

gctgccctcg acaacgctgg taagatcatg agcctcacca agaccgctcc cgattatctg 180

gtcggtcaac agcccgtgga ggacatctcc tccaaccgca tctacaagat tctggagctc 240

aacggctacg acccccagta tgccgcctcc gtcttcctcg gttgggctac caagaagttc 300

ggcaagcgca acactatctg gctcttcggt cccgctacca ctggtaagac caacatcgcc 360

gaagccatcg cccataccgt gcccttttac ggctgcgtca actggaccaa cgagaacttc 420

cccttcaacg actgcgtcga caagatggtc atctggtggg aagagggcaa gatgactgcc 480

aaggtggtcg aatccgccaa ggccattctg ggtggtagca aggtgcgtgt cgaccagaag 540

tgcaagtcct ccgctcagat cgaccccacc cccgtcatcg tgacttccaa caccaacatg 600

tgcgctgtca tcgacggcaa ctccactacc ttcgaacatc agcaacctct gcaagaccgc 660

atgttcaaat tcgagctcac ccgccgtctg gaccacgact tcggcaaagt caccaagcaa 720

gaggtgaagg acttcttccg ttgggccaag gatcacgtcg tggaggtgga gcacgagttc 780

tacgtgaaga agggcggtgc taagaagcgc cccgctccca gcgacgctga tatcagcgag 840

cctaagcgcg tgcgtgagtc cgtcgctcag ccttccacct ccgatgctga agcctccatc 900

aactacgccg accgctacca gaataagtgc agccgccacg tgggcatgaa tctgatgctg 960

ttcccttgcc gccagtgcga gcgcatgaac cagaactcca acatctgctt cacccacggc 1020

cagaaagact gtctggaatg cttccccgtc tccgagtccc aacccgtgtc cgtcgtcaag 1080

aaggcctacc agaagctgtg ctacatccac cacatcatgg gcaaggtgcc cgacgcttgc 1140

actgcttgcg atctcgtgaa cgtggatctg gacgactgca tcttcgagca gtaa 1194

<210> 13

<211> 1194

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> Rep52WT

<400> 13

atggagctgg tcgggtggct cgtggacaag gggattacct cggagaagca gtggatccag 60

gaggaccagg cctcatacat ctccttcaat gcggcctcca actcgcggtc ccaaatcaag 120

gctgccttgg acaatgcggg aaagattatg agcctgacta aaaccgcccc cgactacctg 180

gtgggccagc agcccgtgga ggacatttcc agcaatcgga tttataaaat tttggaacta 240

aacgggtacg atccccaata tgcggcttcc gtctttctgg gatgggccac gaaaaagttc 300

ggcaagagga acaccatctg gctgtttggg cctgcaacta ccgggaagac caacatcgcg 360

gaggccatag cccacactgt gcccttctac gggtgcgtaa actggaccaa tgagaacttt 420

cccttcaacg actgtgtcga caagatggtg atctggtggg aggaggggaa gatgaccgcc 480

aaggtcgtgg agtcggccaa agccattctc ggaggaagca aggtgcgcgt ggaccagaaa 540

tgcaagtcct cggcccagat agacccgact cccgtgatcg tcacctccaa caccaacatg 600

tgcgccgtga ttgacgggaa ctcaacgacc ttcgaacacc agcagccgtt gcaagaccgg 660

atgttcaaat ttgaactcac ccgccgtctg gatcatgact ttgggaaggt caccaagcag 720

gaagtcaaag actttttccg gtgggcaaag gatcacgtgg ttgaggtgga gcatgaattc 780

tacgtcaaaa agggtggagc caagaaaaga cccgccccca gtgacgcaga tataagtgag 840

cccaaacggg tgcgcgagtc agttgcgcag ccatcgacgt cagacgcgga agcttcgatc 900

aactacgcag accgctacca aaacaaatgt tctcgtcacg tgggcatgaa tctgatgctg 960

tttccctgca gacaatgcga gagaatgaat cagaattcaa atatctgctt cactcacgga 1020

cagaaagact gtttagagtg ctttcccgtg tcagaatctc aacccgtttc tgtcgtcaaa 1080

aaggcgtatc agaaactgtg ctacattcat catatcatgg gaaaggtgcc agacgcttgc 1140

actgcctgcg atctggtcaa tgtggatttg gatgactgca tctttgaaca ataa 1194

<210> 14

<211> 8310

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> pFastBac-p10Rep

<400> 14

ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60

ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 420

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 480

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 540

attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 600

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 660

gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 720

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 780

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 840

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 900

agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 960

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 1020

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 1080

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 1140

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 1200

accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 1260

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 1320

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 1380

taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 1440

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1500

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1560

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1620

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1680

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1740

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1800

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1860

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1920

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1980

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 2040

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 2100

acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 2160

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 2220

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 2280

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 2340

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 2400

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 2460

cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2520

tctgtgcggt atttcacacc gcagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2580

agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2640

caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2700

aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2760

cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2820

gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2880

gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2940

ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 3000

ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3060

cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3120

ctcactacgc ggctgctcaa acctgggcag aacgtaagcc gcgagagcgc caacaaccgc 3180

ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3240

atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3300

atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3360

gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3420

gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg 3480

cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3540

aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3600

agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc 3660

gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3720

aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780

cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg ccctggcttc 3840

aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3900

tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3960

atagttctag tggttggcct acgtacccgt agtggctatg gcagggcttg ccgccccgac 4020

gttggctgcg agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga 4080

aacgcgggcg tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg 4140

ggaccgaacc ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt 4200

ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc 4260

tcccccatct cccggtaccg catgctatgc atcagctgct agcttactgc tcgaagatgc 4320

agtcgtccag atccacgttc acgagatcgc aagcagtgca agcgtcgggc accttgccca 4380

tgatgtggtg gatgtagcac agcttctggt aggccttctt gacgacggac acgggttggg 4440

actcggagac ggggaagcat tccagacagt ctttctggcc gtgggtgaag cagatgttgg 4500

agttctggtt catgcgctcg cactggcggc aagggaacag catcagattc atgcccacgt 4560

ggcggctgca cttattctgg tagcggtcgg cgtagttgat ggaggcttca gcatcggagg 4620

tggaaggctg agcgacggac tcacgcacgc gcttaggctc gctgatatca gcgtcgctgg 4680

gagcggggcg cttcttagca ccgcccttct tcacgtagaa ctcgtgctcc acctccacga 4740

cgtgatcctt ggcccaacgg aagaagtcct tcacctcttg cttggtgact ttgccgaagt 4800

cgtggtccag acggcgggtg agctcgaatt tgaacatgcg gtcttgcaga ggttgctgat 4860

gttcgaaggt agtggagttg ccgtcgatga cagcgcacat gttggtgttg gaagtcacga 4920

tgacgggggt ggggtcgatc tgagcggagg acttgcactt ctggtcgaca cgcaccttgc 4980

taccacccag aatggccttg gcggattcga ccaccttggc agtcatcttg ccctcttccc 5040

accagatgac catcttgtcg acgcagtcgt tgaaggggaa gttctcgttg gtccagttga 5100

cgcagccgta aaagggcacg gtatgggcga tggcttcggc gatgttggtc ttaccagtgg 5160

tagcgggacc gaagagccag atagtgttgc gcttgccgaa cttcttggta gcccaaccga 5220

ggaagacgga ggcggcatac tgggggtcgt agccgttgag ctccagaatc ttgtagatgc 5280

ggttggagga gatgtcctcc acgggctgtt gaccgaccag ataatcggga gcggtcttgg 5340

tgaggctcat gatcttacca gcgttgtcga gggcagcctt gatctgggaa cgggagttgc 5400

tggcagcatt gaagctgatg tagctggctt ggtcctcttg gatccactgc ttctcgctag 5460

tgatgccctt gtcgaccagc caaccgacca gttccatggt ggcccgggtt tcggaccgag 5520

atccgcgccc gatggtggga cggtatgaat aatccggaat atttataggt ttttttatta 5580

caaaactgtt acgaaaacag taaaatactt atttatttgc gagatggtta tcattttaat 5640

tatctccatg atctattaat attccggagt atacggacct ttaattcaac ccaacacaat 5700

atattatagt taaataagaa ttattatcaa atcatttgta tattaattaa aatactatac 5760

tgtaaattac attttattta caatcactcg acgaagactt gatcagcggc cgccaccatg 5820

gcggggtttt acgagattgt gattaaggtc cccagcgacc ttgacgagca tctgcccggc 5880

atttctgaca gctttgtgaa ctgggtggcc gagaaggaat gggagttgcc gccagattct 5940

gacatggatc tgaatctgat tgagcaggca cccctgaccg tggccgagaa gctgcagcgc 6000

gactttctga cggaatggcg ccgtgtgagt aaggccccgg aggccctttt ctttgtgcaa 6060

tttgagaagg gagagagcta cttccacatg cacgtgctcg tggaaaccac cggggtgaaa 6120

tccatggttt tgggacgttt cctgagtcag attcgcgaaa aactgattca gagaatttac 6180

cgcgggatcg agccgacttt gccaaactgg ttcgcggtca caaagaccag aaatggcgcc 6240

ggaggcggga acaaggtggt ggatgagtgc tacatcccca attacttgct ccccaaaacc 6300

cagcctgagc tccagtgggc gtggactaat atggaacagt atttaagcgc ctgtttgaat 6360

ctcacggagc gtaaacggtt ggtggcgcag catctgacgc acgtgtcgca gacgcaggag 6420

cagaacaaag agaatcagaa tcccaattct gatgcgccgg tgatcagatc aaaaacttca 6480

gccaggtaca tggagctggt cgggtggctc gtggacaagg ggattacctc ggagaagcag 6540

tggatccagg aggaccaggc ctcatacatc tccttcaatg cggcctccaa ctcgcggtcc 6600

caaatcaagg ctgccttgga caatgcggga aagattatga gcctgactaa aaccgccccc 6660

gactacctgg tgggccagca gcccgtggag gacatttcca gcaatcggat ttataaaatt 6720

ttggaactaa acgggtacga tccccaatat gcggcttccg tctttctggg atgggccacg 6780

aaaaagttcg gcaagaggaa caccatctgg ctgtttgggc ctgcaactac cgggaagacc 6840

aacatcgcgg aggccatagc ccacactgtg cccttctacg ggtgcgtaaa ctggaccaat 6900

gagaactttc ccttcaacga ctgtgtcgac aagatggtga tctggtggga ggaggggaag 6960

atgaccgcca aggtcgtgga gtcggccaaa gccattctcg gaggaagcaa ggtgcgcgtg 7020

gaccagaaat gcaagtcctc ggcccagata gacccgactc ccgtgatcgt cacctccaac 7080

accaacatgt gcgccgtgat tgacgggaac tcaacgacct tcgaacacca gcagccgttg 7140

caagaccgga tgttcaaatt tgaactcacc cgccgtctgg atcatgactt tgggaaggtc 7200

accaagcagg aagtcaaaga ctttttccgg tgggcaaagg atcacgtggt tgaggtggag 7260

catgaattct acgtcaaaaa gggtggagcc aagaaaagac ccgcccccag tgacgcagat 7320

ataagtgagc ccaaacgggt gcgcgagtca gttgcgcagc catcgacgtc agacgcggaa 7380

gcttcgatca actacgcaga caggtaccaa aacaaatgtt ctcgtcacgt gggcatgaat 7440

ctgatgctgt ttccctgcag acaatgcgag agaatgaatc agaattcaaa tatctgcttc 7500

actcacggac agaaagactg tttagagtgc tttcccgtgt cagaatctca acccgtttct 7560

gtcgtcaaaa aggcgtatca gaaactgtgc tacattcatc atatcatggg aaaggtgcca 7620

gacgcttgca ctgcctgcga tctggtcaat gtggatttgg atgactgcat ctttgaacaa 7680

taaatgattt aaatcaggta tggctgccga tggttatctt ccagattggc tcgaggacac 7740

tctctctgat ctagagcctg cagtctcgac aagcttgtcg agaagtacta gaggatcata 7800

atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 7860

ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 7920

aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 7980

cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatctgatca 8040

ctgcttgagc ctaggagatc cgaaccagat aagtgaaatc tagttccaaa ctattttgtc 8100

atttttaatt ttcgtattag cttacgacgc tacacccagt tcccatctat tttgtcactc 8160

ttccctaaat aatccttaaa aactccattt ccacccctcc cagttcccaa ctattttgtc 8220

cgcccacagc ggggcatttt tcttcctgtt atgtttttaa tcaaacatcc tgccaactcc 8280

atgtgacaaa ccgtcatctt cggctacttt 8310

<210> 15

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> P1

<400> 15

gatccggtac cacgcgtcta g 21

<210> 16

<211> 26

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> P2

<400> 16

ctcgacgtcg actttacttg tacagc 26

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> P3

<400> 17

gcggggtttt acgagattgt g 21

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> P4

<400> 18

ggggtgcctg ctcaatcaga 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> P5

<400> 19

gcagcacaca ctgacatcca 20

<210> 20

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> P6

<400> 20

gatcaccggc gcatcagaat tg 22

<210> 21

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> P7

<400> 21

acttcaagat ccgccacaac at 22

<210> 22

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> P8

<400> 22

tctcgttggg gtcttgctca g 21

<210> 23

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> M13 F

<400> 23

cccagtcacg acgttgtaaa acg 23

<210> 24

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> M13 R

<400> 24

agcggataac aatttcacac agg 23

Claims

1. An isolated nucleic acid molecule comprising, in order, a first polyA, a nucleotide sequence encoding a Rep78 protein, a first promoter, a second promoter, a protein nucleotide sequence encoding a Rep52 protein, and a second polyA, wherein the first promoter is a nucleotide sequence encoding a Rep78 protein and a transcriptional promoter of the first polyA, the second promoter is a nucleotide sequence encoding a Rep52 protein and a transcriptional promoter of the second polyA, wherein the nucleotide sequence encoding a Rep52 protein and/or the sequence of the nucleotide sequence encoding a Rep78 protein is codon optimized to avoid homologous recombination, the first promoter is a p10 promoter, the second promoter is a polh promoter, the nucleotide sequence encoding a Rep78 protein is as shown in SEQ ID No. 11, and the nucleotide sequence encoding a Rep52 protein is as shown in SEQ ID No. 12.

2. The isolated nucleic acid molecule of claim 1, wherein the p10 promoter comprises the nucleotide sequence set forth in SEQ ID No. 9.

3. The isolated nucleic acid molecule of claim 1, wherein the polh promoter comprises the nucleotide sequence set forth in SEQ ID No. 10.

4. The isolated nucleic acid molecule of claim 1, wherein the 5 'end of the first promoter is directly or indirectly linked to the 5' end of the second promoter.

5. The isolated nucleic acid molecule of claim 1, wherein the 3 'end of the first promoter is directly or indirectly linked to the 5' end of the nucleotide sequence encoding Rep 78.

6. The isolated nucleic acid molecule of claim 1, wherein the 3 'end of the nucleotide sequence encoding the Rep78 protein is directly or indirectly linked to the 5' end of the first polyA.

7. The isolated nucleic acid molecule of claim 1, wherein the 3 'end of the second promoter is directly or indirectly linked to the 5' end of the nucleotide sequence encoding Rep 52.

8. The isolated nucleic acid molecule of claim 1, wherein the 3 'end of the nucleotide sequence encoding the Rep52 protein is directly or indirectly linked to the 5' end of the second polyA encoding.

9. The isolated nucleic acid molecule of claim 1, wherein the polyA is selected from the group consisting of: any one of SV40 polyA and HSV TK polyA.

10. The isolated nucleic acid molecule of claim 1 comprising the nucleotide sequence set forth in SEQ ID No. 8.

11. A vector comprising the isolated nucleic acid molecule of any one of claims 1-10.

12. The vector of claim 11, which is a viral vector.

13. The vector of claim 11, which is a baculovirus vector.

14. The vector of claim 11, which is a pFastBac vector.

15. The vector according to any one of claims 11-14, comprising the nucleotide sequence shown in SEQ ID No. 14.

16. A cell comprising the isolated nucleic acid molecule of any one of claims 1-10 or the vector of any one of claims 11-15, the cell being of a non-plant or animal variety.

17. The cell of claim 16, which is an insect cell.

18. The cell of claim 16, which is Spodoptera frugiperda cells.

19. A baculovirus expression system comprising a first baculovirus vector and a second baculovirus vector comprising a nucleic acid sequence encoding a gene of interest, said first baculovirus vector being a baculovirus vector as defined in any one of claims 13-14.

20. The baculovirus expression system of claim 19, from 5 'end to 3' end, the nucleic acid sequence encoding the gene of interest comprising, in order, an inverted terminal repeat (INVERTED TERMINAL REPEAT, ITR) of the first parvovirus, the gene of interest and the second ITR.

21. The baculovirus expression system of any one of claims 20, wherein the first ITR further comprises at least one promoter between the first ITR and the gene of interest.

22. The baculovirus expression system of claim 20, wherein the first ITR further comprises at least one eukaryotic promoter between the first ITR and the gene of interest.

23. The baculovirus expression system of claim 20, wherein the first ITR further comprises at least one mammalian cell promoter between the first ITR and the gene of interest.

24. The baculovirus expression system of claim 20, wherein the first ITR further comprises a mammalian cell promoter and an insect cell promoter between the first ITR and the gene of interest.

25. The baculovirus expression system of claim 24 wherein the mammalian cell promoter is selected from the group consisting of a broad range promoter and a tissue specific promoter.

26. The baculovirus expression system of claim 25, wherein the broad-spectrum promoter is a CMV, SV40, EF1a, CAG or UBC promoter.

27. The baculovirus expression system of claim 25, wherein the tissue specific promoter is ALB, hAAT, TBG, TTR, GFAP, MHCK7 or hSyn promoter.

28. The baculovirus expression system of claim 24 wherein the insect cell promoter is a p10 promoter.

29. The baculovirus expression system of claim 24, the mammalian promoter and the insect cell promoter being CMV and p10 promoters.

30. An insect cell comprising the baculovirus expression system of any one of claims 19-29.

31. The insect cell of claim 30, further comprising a third nucleotide sequence comprising two parvoviral ITR nucleotide sequences and at least one nucleotide sequence encoding a gene of interest, and wherein the at least one nucleotide sequence encoding a gene of interest is located between the two parvoviral ITR nucleotide sequences.

32. The insect cell of claim 31, wherein the parvovirus is an adeno-associated virus.

33. The insect cell of claim 31, wherein the third nucleotide sequence is part of another nucleic acid construct, wherein each nucleotide sequence encoding a gene of interest is operably linked to an expression control sequence for mammalian expression.

34. The insect cell of claim 33, wherein the nucleic acid construct is an insect cell compatible vector.

35. The insect cell of claim 33, wherein the nucleic acid construct is a baculovirus vector.

36. Use of a baculovirus expression system as defined in any one of claims 19-29 or an insect cell as defined in any one of claims 30-35 in the preparation of a nucleic acid molecule of interest.

37. The use of claim 36, wherein the nucleic acid molecule of interest is a linear DNA molecule (neDNA) having a covalently closed end.

38. A method of producing a nucleic acid molecule of interest comprising culturing the insect cell of any one of claims 30-35.

39. The method of manufacturing according to claim 38, comprising:

a) Providing a baculovirus expression system of any one of claims 19-29;

b) Inserting a gene sequence of interest into said second baculovirus vector;

c) Co-transfecting the first baculovirus vector and the second baculovirus vector into an insect cell;

d) Growing the insect cell under conditions that allow replication and release of DNA comprising the gene of interest;

e) Collecting the nucleic acid molecule of interest.

40. The method of claim 39, wherein isolating the nucleic acid molecule of interest is also protected.

41. A kit comprising the isolated nucleic acid molecule of any one of claims 1-10, the baculovirus expression system of any one of claims 19-29, or the insect cell of any one of claims 30-35.