CN115369098A - A novel CRISPR-associated transposase - Google Patents

Abstract

The invention discloses a novel CRISPR-related transposase, which is derived from Pseudoalteromonas translucent KMM520, can recognize 16 kinds of PAMs, and can insert cargo genes into 8 sites at one time, with an efficiency of 100%, which is higher than that of the source CRISPR-associated transposase from Vibrio cholerae Tn6677; the efficiency of transposing the 15.4kb cargo gene to the corresponding target site is 100%, and can be used in the same E. coli with the CRISPR-associated transposase from Vibrio cholerae Tn6677, and They can function without interfering with each other, and have broad application prospects.

Description

A novel CRISPR-associated transposase

technical field

The invention belongs to the technical field of gene editing, and in particular relates to a novel CRISPR-related transposase and its application in gene editing.

Background technique

In metabolic engineering, by adjusting the dosage and ratio of enzyme genes, different enzymes in metabolic pathways or different metabolic pathways can be coordinated to maximize the overall reaction speed. The most common means of increasing the dosage of gene expression cassettes in bacteria are plasmids, but suffer from genetic stability issues. Individual integration of gene expression cassettes into bacterial chromosomes is time-consuming, making rapid testing of many constructions difficult. Although CRISPR-Cas can cut multiple copies of the genome or simultaneously target multiple different sequences with crRNA arrays, it is limited by the repair efficiency of double-strand breaks and it is difficult to insert more than 3 copies of gene expression cassettes at one time. Therefore, a better way to obtain combinations of different gene dosages is needed.

In 2019, the Zhang Feng research group of the Broad Institute and the Sam Sternberg research group of Columbia University announced two similar research results: using bacterial transposable genes to precisely insert DNA sequences into the genome without cutting DNA. Among them, Zhang Feng's research group extracted a transposase from cyanobacteria and named it CAST, which stands for CRISPR-associated transposase (CRISPR-associated transposase). After discovering a unique transposable gene in Vibrio cholerae, the Sternberg research group developed a gene called INTEGRATE (Insertion of transposable elements by guide RNA-assisted targeting, guide RNA-assisted targeted transposable element insertion) Editing tools that can insert large stretches of genes in the genome without introducing DNA breaks. The new technology, INTEGRATE, utilizes transposable genes to insert DNA sequences into the genome without cutting the DNA. Both the CAST system and the INTEGRATE system can integrate DNA fragments into preset sites on the E. coli chromosome through transposition without relying on homologous recombination, without residual resistance markers and double-stranded DNA breaks.

Based on the CAST system and the INTEGRATE system, the inventor developed a multi-copy gene insertion system MUCICAT (see patent document CN202010083919.7), which can obtain a strain with 10 copies of the cargo gene (Cargo gene) inserted into the chromosome in 5 days, with Editable, fast, marker-free, fixed-point and other advantages, MUCICAT has been successfully applied to the construction of enzyme engineering strains and metabolic engineering strains (Zhang, Y., Multicopy chromosome integration using CRISPR-associated transposases.Acs Synthetic Biology, 2020.). However, metabolic engineering and synthetic biology usually involve optimization of the expression levels of multiple enzymes or pathways. In a round of transposition, MUCICAT based on a single CRISPR-related transposase can only explore the optimal dosage of a single cargo gene carrying a single or multiple genes, and cannot screen the optimal ratio of gene dosages for multiple genes or pathways. There are no reports of chromosomal copy number techniques for parallel amplification of multiple genes/pathways. MUCICAT technology containing multiple CRISPR-related transposases that are orthogonal to each other is expected to achieve simultaneous and independent amplification of multiple genes or pathways in a single cell, forming a strain library with different gene dosage ratios to screen the optimal multiple genes or pathways dosage ratio.

Among the CRISPR transposases with known activity, only the I-F3 type CRISPR transposase derived from Vibrio cholerae Tn6677 had high insertion efficiency and on-target rate (Klompe, S.E., et al., Transposon-encoded CRISPR–Cas systemsdirect RNA-guided DNA integration.Nature, 2019.571(7764):p.219-225.), derived from the V-K type of Hofmannis sp. and Anabaena cylindrica (Jonathan Strecker et al., RNA-guided DNAinsertion with CRISPR-associated transposases.Science, 2019), I-F3 types of Aeromonas salmonicida S44 (Michael T.Petassi et al., Guide RNA Categorization Enables Target SiteChoice in Tn7-CRISPR-Cas Transposons.Cell, 2020), changeable fish Both types I-B of Anabaena and Peltigeramembranacea cyanobiont 210A have low efficiency or/and high off-target rate (Makoto Saito, A.L. and Jonathan Strecker, Han Altae-Tran, Rhiannon K. Macrae, Feng Zhang, Dual modes of CRISPR-associated transposon homing. Cell, 2021 .).

Contents of the invention

According to the characteristics of the existing CRISPR transposases, ideally, if there are other new and efficient CRISPR-related transposases, or an orthogonal CRISPR-related transposase system can be formed with MUCICAT, it can be applied to complex metabolic engineering and synthesis in biological design. In order to achieve this goal, the inventors conducted extensive screening of CRISPR transposases from other microorganisms, and found a type I-F3 CRISPR transposition system derived from Pseudoalteromonas translucida KMM520, which was found in The insertion efficiency in Escherichia coli is no less than or even better than that of Vibrio cholerae Tn6677, and can be inserted into the eda-purT and lacZ sites without interfering with Vibrio cholerae Tn6677, respectively. This novel CRISPR transposition system recognizes all 16 PAMs without PAM dependence.

Therefore, the first aspect of the present invention is to provide a CRISPR-related transposase, which includes a polypeptide selected from the group consisting of the transposase protein tnsA derived from Pseudoalteromonas bacteria, the transposase protein tnsA derived from Pseudoalteromonas Transposase protein tnsB from bacteria of the genus Pseudoalteromonas, transposase protein tnsC from bacteria of the genus Pseudoalteromonas, transposase protein tniQ from bacteria of the genus Pseudoalteromonas, Bacterial nuclease protein Cas5/8, nuclease protein Cas6 derived from bacteria of the genus Pseudoalteromonas, nuclease protein Cas7 derived from bacteria of the genus Pseudoalteromonas.

Preferably, the bacteria of the genus Pseudoalteromonas is Pseudoalteromonas translucidum. More preferably, the Pseudoalteromonas translucida is Pseudoalteromonas translucida KMM520.

In a specific embodiment, the above-mentioned CRISPR-related transposase preferably includes a polypeptide selected from the group consisting of:

tnsA: it is a polypeptide having the amino acid sequence of SEQ ID NO: 1, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO: 1, and Functionally identical polypeptides;

tnsB: it is a polypeptide having the amino acid sequence of SEQ ID NO: 2, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO: 2, and Functionally identical polypeptides;

tnsC: it is a polypeptide having the amino acid sequence of SEQ ID NO:3, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO:3, and Functionally identical polypeptides;

tniQ: it is a polypeptide having the amino acid sequence of SEQ ID NO:4, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO:4, and Functionally identical polypeptides;

Cas5/8: It is a polypeptide having the amino acid sequence of SEQ ID NO:5, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO:5 , and a polypeptide with the same function;

Cas6: it is a polypeptide having the amino acid sequence of SEQ ID NO:6, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO:6, and Functionally identical polypeptides;

Cas7: it is a polypeptide having the amino acid sequence of SEQ ID NO: 7, or has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO: 7, and functionally identical peptides.

The above-mentioned nucleases Cas5/8, Cas7, and Cas6 are the Cascade complexes of the type I CRISPR system, which are associated with the transposases tnsABC and tniQ to become CRISPR-associated transposases. These polypeptides are all derived from Pseudoalteromonas translucida KMM520 (Pseudoalteromonas translucida KMM520).

The second aspect of the present invention provides a gene encoding the above-mentioned polypeptide.

In a specific embodiment, the gene encoding the polypeptide tnsA having the amino acid sequence of SEQ ID NO: 1 is the nucleotide sequence of SEQ ID NO: 8, or has more than 80% homology with SEQ ID NO: 8, preferably Polynucleotides with more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology;

The gene encoding the polypeptide tnsB having the amino acid sequence of SEQ ID NO: 2 is the nucleotide sequence of SEQ ID NO: 9, or has more than 80% homology, preferably more than 85% homology, more Polynucleotides with preferably more than 90% homology, more preferably more than 95% homology;

The gene encoding the polypeptide tnsC having the amino acid sequence of SEQ ID NO:3 is the nucleotide sequence of SEQ ID NO:10, or has more than 80% homology, preferably more than 85% homology, more homology with SEQ ID NO:10 Polynucleotides with preferably more than 90% homology, more preferably more than 95% homology;

The gene encoding the polypeptide tniQ having the amino acid sequence of SEQ ID NO: 4 is the nucleotide sequence of SEQ ID NO: 11, or has more than 80% homology, preferably more than 85% homology, more Polynucleotides with preferably more than 90% homology, more preferably more than 95% homology;

The gene encoding the polypeptide Cas5/8 having the amino acid sequence of SEQ ID NO:5 is the nucleotide sequence of SEQ ID NO:12, or has more than 80% homology, preferably more than 85% homology with SEQ ID NO:12 , more preferably a polynucleotide with more than 90% homology, more preferably more than 95% homology;

The gene encoding the polypeptide Cas6 having the amino acid sequence of SEQ ID NO: 6 is the nucleotide sequence SEQ ID NO: 13, or has more than 80% homology, preferably more than 85% homology, more homology with SEQ ID NO: 13 Polynucleotides with preferably more than 90% homology, more preferably more than 95% homology;

The gene encoding the polypeptide Cas7 having the amino acid sequence of SEQ ID NO: 7 is the nucleotide sequence SEQ ID NO: 14, or has more than 80% homology, preferably more than 85% homology, more homology with SEQ ID NO: 14 It is preferably a polynucleotide with a homology of more than 90%, more preferably a homology of more than 95%.

The third aspect of the present invention is to provide a CRISPR transposon system. Like the transposition system INTEGRATE system or CAST system, the CRISPR transposon system of the present invention also includes plasmid pQCascade, helper plasmid pTns, and helper plasmid pDonor carrying cargo genes. Any two of these three plasmids can even be combined into one plasmid, and even these three plasmids can be combined together to form one plasmid. in particular,

A plasmid pQCascade for the CRISPR transposon system, which includes a gene segment selected from the group consisting of: the above-mentioned Cas5/8 coding gene; the above-mentioned Cas6 coding gene; the above-mentioned Cas7 coding gene; the above-mentioned tniQ coding gene.

Since the above polypeptides are all derived from Pseudoalteromonas translucida KMM520 ( Pseudoalteromonas tr anslucida KMM520), the plasmid pQCascade is named pQCascadePtr herein. Similarly, the plasmids pTns and pDonor of the present invention may be denoted as pTnsPtr and pDonorPtr, respectively, hereinafter.

Preferably, the above-mentioned plasmid pQCascadePtr may also include the following genes: crRNA sequence targeting the target site in the genome, CloDF13 replicon, promoter such as anhydrocycline-inducible promoter, streptomycin resistance gene.

The crRNA in the above CRISPR transposase can function in the form of an array.

In one embodiment, the above-mentioned spacer of the crRNA series may be a spacer targeting a single site in the genome of the cell to be treated.

In another embodiment, the above-mentioned crRNA series may be a crRNA array (also known as CRISPRarray, crRNA array or array) targeting multiple sites in the genome of the cell to be treated.

Preferably, the above-mentioned crRNA sequence is a crRNA array targeting multiple sites in the genome, wherein the repeat sequence region repeat includes more than one sequence selected from the following group: the nucleotide sequence is repeat1 of SEQ ID NO: 15, nucleotide The sequence is repeat2 of SEQ ID NO: 16, the nucleotide sequence is repeat3 of SEQ ID NO: 17, the nucleotide sequence is repeat4 of SEQ ID NO: 18, and these nucleotide sequences are 32 in SEQ ID NOs: 15-18 Each N (ie [N32]) is any base A, T, G or C.

A helper plasmid pTnsPtr for the CRISPR transposon system, which is used in conjunction with the above-mentioned plasmid pQCascadePtr, which includes a gene segment selected from the group consisting of the above-mentioned tnsA-encoding gene, the above-mentioned tnsB-encoding gene, and the above-mentioned tnsC-encoding gene .

When the above-mentioned CRISPR transposase gene sequence is used for CRISPR transposition, it needs to be combined with the host (gram-negative bacteria such as Escherichia coli, natrivibrio, and Tatumella citronella; gram-positive bacteria such as glutamic acid rod Bacillus) available promoters, Leftend (LE)-cargo-Right end (RE) work together, either in the form of a plasmid or in an integrated form.

Preferably, the above-mentioned helper plasmid pTnsPtr also includes the following genes: ColA replicon, promoter such as anhydrocycline-inducible promoter, and kanamycin resistance gene.

As an embodiment in which two plasmids are combined into one plasmid, the present invention provides a plasmid pQCasTnsPtr for the CRISPR transposon system, which is formed by merging the above-mentioned plasmid pQCascadePtr and the helper plasmid pTnsPtr, including: the above-mentioned Cas5 /8, Cas6, Cas7, tniQ, tnsA, tnsB, and tnsC genes, crRNA sequences targeting genomic target sites, ColA replicons, promoters such as anhydrocycline-inducible promoters, kanamycin resistance genes.

A helper plasmid pDonorPtr for the CRISPR transposon system, which is used in conjunction with the above-mentioned plasmid pQCascadePtr and the above-mentioned helper plasmid pTnsPtr, which includes a gene segment selected from the group: sequence Leftend (LE), its nucleotide sequence It is SEQ ID NO:19, or has more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO:19 And comprise the 3' end 33bp sequence of SEQ ID NO:19; Sequence Right end (RE), its nucleotide sequence is SEQ ID NO:20, or there is more than 80% homology with SEQ ID NO:20, preferably More than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology and containing the 27bp sequence of the 5' end of SEQ ID NO: 20; the target cargo gene (Cargo gene).

Since the LE with the nucleotide sequence of SEQ ID NO: 19 and the RE with the nucleotide sequence of SEQ ID NO: 20 are derived from Pseudoalteromonas translucidus KMM520 ( P seudoalteromonas tr anslucida KMM520), the plasmid pDonor Name it pDonorPtr.

Preferably, the above-mentioned auxiliary plasmid pDonorPtr can also include the following genes: pMB1 replicon, ampicillin resistance gene, target cargo gene (Cargo gene); or the following genes: p15A replicon, chloramphenicol resistance gene, target cargo Gene (Cargo gene).

As an embodiment in which the above three plasmids are combined into one plasmid, the present invention provides a plasmid pEffectorPtr for the CRISPR transposon system, which is formed by merging the above-mentioned plasmid pQCascadePtr, the above-mentioned helper plasmid pTnsPtr and the above-mentioned helper plasmid pDonorPtr components, including: the above-mentioned Cas5/8, Cas6, Cas7, tniQ, tnsA, tnsB and tnsC genes, Left end (LE) and Right end (RE) sequences, target cargo genes, crRNA sequences targeting genomic target sites, ColA replicon, promoters such as anhydrocycline-inducible promoters, kanamycin resistance genes.

Based on the above plasmids, the fourth aspect of the present invention provides a CRISPR transposon system, which includes: plasmids pQCascadePtr, pTnsPtr and pDonorPtr; or plasmids pQCasTnsPtr and pDonorPtr; or plasmid pEffectorPtr.

A fifth aspect of the present invention provides a combination of the above-mentioned CRISPR transposon system derived from Pseudomonas translucidus KMM520 and the prior art CRISPR transposon system derived from Vibrio cholerae (Vibrio cholerae) Tn6677 Combinations use, specifically,

A CRISPR transposon system, which, in addition to including the above-mentioned CRISPR transposon system (including: plasmids pQCascadePtr, pTnsPtr and pDonorPtr; or plasmids pQCasTnsPtr and pDonorPtr; or plasmid pEffectorPtr), also includes Vibrio cholerae ( Vibrio ch olerae) Tn6677-derived CRISPR transposase-associated plasmid.

The gene sequence of the type I-F3 CRISPR transposase of Vibrio cholerae Tn6677 is NCBI: NZ_ALED01000027.1.

The above-mentioned CRISPR transposase-related plasmids derived from Vibrio cholerae Tn6677 include plasmid pQCasTnsVch and helper plasmid pDonorVch, wherein

Plasmid pQCasTnsVch includes: Cas5/8, Cas6, Cas7, tniQ, tnsA, tnsB and tnsC genes derived from Vibrio cholerae Tn6677, CloDF13 replicator, promoter such as anhydrocycline-inducible promoter, streptomycin resistance gene;

Plasmid pDonorVch includes CRISPR array derived from Vibrio cholerae Tn6677, Left end (LE) and Right end (RE), pMB1 replicon, ampicillin resistance gene, target cargo gene (Cargo gene).

In one embodiment, similar to the combination of two or three plasmids of the present invention into one plasmid pQCasTnsPtr and pEffectorPtr, the above-mentioned plasmids pQCasTnsVch and pDonorVch can be combined into plasmid pEffectorVch.

In the case that the above-mentioned promoter adopts anhydrotetracycline-inducible promoter, when the above-mentioned CRISPR transposon system derived from Pseudomonas translucidum KMM520 is used to transform Escherichia coli BL21 (DE3), BL21Star ^TM (DE3) or W3110 (DE3) and other strains can be induced with anhydrotetracycline. That is, when the plasmids pQCasTnsPtr and pDonorPtr are used to transform strains such as Escherichia coli BL21(DE3), BL21Star ^™ (DE3) or W3110(DE3), anhydrotetracycline is used for induction.

Similarly, when the above-mentioned promoter adopts anhydrocycline-inducible promoter, when the above-mentioned CRISPR transposon system derived from Pseudomonas translucidus KMM520 is combined with the CRISPR transposon system derived from Vibrio cholerae Tn6677 When used together, anhydrocycline can also be used for induction. That is, when plasmids pEffectorPtr and pEffectorVch, or plasmids pQCasTnsVch, pDonorVch, pQCasTnsPtr, and pDonorPtr are used to transform strains such as Escherichia coli BL21 (DE3), BL21Star ^TM (DE3) or W3110 (DE3), anhydrotetracycline is used for induction.

These two microbial-derived CRISPR transposon systems can be used in the same Escherichia coli, and can function without interfering with each other, and the two function orthogonally.

The sixth aspect of the present invention provides the above-mentioned CRISPR-related transposase, the above-mentioned gene encoding a polypeptide, the above-mentioned plasmid pQCascadePtr, the above-mentioned plasmid pTnsPtr, the plasmid pQCasTnsPtr, the plasmid pDonorPtr, the plasmid pEffectorPtr, the above-mentioned CRISPR transposon system Applications in gene editing.

The gene editing can be gene editing in any cell, especially gene editing in microorganism (including fungi and bacteria) cells, especially gene editing in industrial microorganism cells.

Preferably, the bacteria for gene editing include Gram-negative bacteria such as Escherichia coli, Narvibrio natrium, Tatumella citrum, Gram-positive bacteria such as Corynebacterium glutamicum, etc., but are not limited thereto.

Experiments have proved that the new CRISPR-associated transposase system developed by the present invention can insert cargo genes into 8 sites at one time, with an efficiency of 100%, which is higher than that of the CRISPR-associated transposase derived from Vibrio cholerae Tn6677; transposition 15.4kb The efficiency of the cargo gene to the corresponding target is 100%; the novel CRISPR-associated transposase can recognize 16 PAMs. In the present invention, two CRISPR-related transposase systems are used in the same Escherichia coli for the first time, and they can function without interfering with each other, thus providing an option for accelerating the construction of metabolic engineering strains.

Description of drawings

Fig. 1 has shown the gel electrophoresis photograph of targeting crRNA3 (lacZ) site in embodiment 1.

Fig. 2 shows the gel electrophoresis of the cargo gene GFP insertion situation at 8 sites in the genome of the strain in Example 2. Where NC is a negative control (Negative Control).

Fig. 3 is a statistical bar chart of the 8-site insertion status of each cloned genome verified by colony PCR and nucleic acid gel electrophoresis in Example 2. The abscissa is the copy number of the cargo gene GFP, and the ordinate is the insertion rate of the cargo gene GFP.

Figure 4 shows the gel electrophoresis images of the insertion of the "terminator sequence" of the cargo gene GFP carried by Ptr and the cargo gene "terminator sequence" carried by Vch at 6 sites in the genome of the strain in Example 3. where NC is the negative control.

Fig. 5 is a schematic diagram of the structure of plasmid pQCascadePtr.

Fig. 6 is a schematic diagram of the structure of plasmid pDonorPtr.

Fig. 7 is a schematic diagram of the structure of plasmid pTnsPtr.

Fig. 8 is a schematic diagram of the structure of plasmid pQCasTnsPtr.

Fig. 9 is a schematic diagram of the structure of plasmid pQCasTnsVch.

Fig. 10 is a schematic diagram of the structure of plasmid pEffectorPtr.

Fig. 11 is a schematic diagram of the structure of plasmid pEffectorVch.

Fig. 12 is a schematic diagram of the structure of plasmid pVnQCasTnsPtr.

Fig. 13 is a schematic diagram of the structure of plasmid pCgQCasTnsVch.

Fig. 14 is a schematic diagram of the structure of plasmid pCgDonorPtr.

Fig. 15 shows the photograph of the gel electrophoresis image targeting the crtYf gene locus of the Corynebacterium glutamicum ATCC13032 strain in Example 5.

Detailed ways

The novel CRISPR-associated transposase system of the present invention is derived from Pseudoalteromonas translucenta KMM520, which is a further development and improvement of the gene editing tools CAST system, INTEGRATE system and MUCICAT system, especially the gene copy amount and cargo gene insertion efficiency improvement.

In this paper, for simplicity of description, the term "CRISPR-associated transposase system" is sometimes abbreviated as "CRISPR-associated transposase", "CRISPR transposase", or "CRISPR transposase system", etc., which mean the same can be used interchangeably.

In this paper, for the sake of simplicity of description, sometimes a certain protein such as Cas6 and the name of its encoding gene (DNA) are mixed, and those skilled in the art should understand that they represent different substances in different description occasions. Their meanings are easily understood by those skilled in the art depending on the context and context. For example, when tnsA is used to describe the function or class of transposase, it refers to the protein; when described as a gene, it refers to the gene encoding the transposase tnsA protein.

Similarly, for simplicity of description, sometimes RNA such as crRNA and the name of its coding gene are mixed, and those skilled in the art should understand that they represent different substances in different description occasions. Their meanings are easily understood by those skilled in the art depending on the context and context.

Each of the plasmids pQCascadePtr, pTnsPtr, pDonorPtr, pQCasTnsPtr, and pEffectorPtr provided by the present invention contains multiple genetic elements, for example, the plasmid pQCascadePtr contains Cas5/8 gene, Cas6 gene, Cas7 gene, tniQ coding gene, crRNA gene, CloDF13 replication promoters, promoters such as anhydrocycline-inducible promoters, and streptomycin resistance genes, the sequence of these gene elements can be arbitrary, and those skilled in the art can arrange them according to their habits and easily prepare plasmids.

It should be understood that for the coding genes of the polypeptides Cas5/8, Cas7, Cas6, tnsABC (i.e. tnsA, tnsB and tnsC) and tniQ of the present invention, those skilled in the art can perform codon optimization according to the specific species of the cells to be treated, such as Escherichia coli , but not limited to the above-mentioned nucleotide sequence SEQ ID NOs:8-14.

The purpose of codon optimization is to enable optimal expression of these polypeptides in the cells to be treated. Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms often show a particular preference for one of several codons encoding the same amino acid due to mutation propensity and natural selection. For example, in fast-growing microorganisms such as E. coli, codons are optimized to reflect the composition of their respective genomic tRNA pools. Thus, in fast-growing microorganisms, low-frequency codons for amino acids can be replaced by high-frequency codons for the same amino acids. Thus, expression of optimized DNA sequences is improved in fast growing microorganisms.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are for the purpose of illustration only and not limitation of the present invention. In addition, it should be understood that after reading the concept of the present invention, various changes or adjustments made by those skilled in the art should fall within the protection scope of the present invention, and these equivalent forms also belong to the appended claims of the present application The scope of the book is limited.

This article involves the addition amount, content and concentration of various substances, and the percentage content mentioned therein refers to the mass percentage content unless otherwise specified.

In the examples herein, if there is no specific statement about the reaction temperature or operating temperature, the temperature usually refers to room temperature (15-30° C.).

Example

Materials and methods

The whole gene synthesis in the examples was completed by Nanjing GenScript Biotechnology Co., Ltd., the primer synthesis was completed by Bosun Biotechnology (Shanghai) Co., Ltd., and the sequencing was completed by Qingke Biotechnology Co., Ltd.

The molecular biology experiments in the examples include plasmid construction, enzyme digestion, connection, competent cell preparation, transformation, medium preparation, etc., mainly referring to "Molecular Cloning Experiment Guide" (third edition), J. Sambrook, Edited by D.W. Russell (US), translated by Huang Peitang et al., Science Press, Beijing, 2002). The specific experimental conditions can be determined by simple experiments if necessary.

PCR amplification experiments were carried out according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers. It can be adjusted by simple experiment if necessary.

LB medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH7.2, sterilized under high temperature and high pressure at 121°C for 20min. Add 20g/L agar powder to the solid medium.

LBv2 medium: add v2 salt (21.7204g mmol/L NaCl, 0.34.2g mmol/LKCl, 4.723.14g mmol/L MgCl ₂ ) to LB medium.

BHIS medium: 37g/L BHI, 91g/L sorbitol. Add 20g/L agar powder to the solid medium.

Plasmids pQCascadePtr, pTnsPtr, and pCgQCasTnsPtr used in the examples were commissioned by Nanjing GenScript Biotechnology Co., Ltd. to construct and synthesize them. Any unit or individual can obtain these plasmids to verify the present invention, but they cannot be used for other purposes, including development and utilization, scientific research and teaching, without the permission of the Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences. in

Plasmid pQCascadePtr comprises genes tniQ (SEQ ID NO:11), Cas5/8 (SEQ ID NO:12), Cas7 (SEQ ID NO:14) and Cas6 (SEQ ID NO:13) derived from Pseudoalteromonas translucida KMM520, targeting The crRNA sequence of the genomic target site, CloDF13 replicon, promoter such as anhydrocycline-inducible promoter, streptomycin resistance gene was commissioned to synthesize by Nanjing GenScript Biotechnology Co., Ltd., and its nucleotide sequence is SEQ ID NO: 21 , whose structure is shown in Figure 5.

Plasmid pDonorPtr contains gene LE (SEQ ID NO:19) and RE (SEQ ID NO:20) sequences derived from Pseudoalteromonas translucida KMM520, pMB1 replicon, ampicillin resistance gene, target cargo gene Cargo such as chloramphenicol without promoter The protein-resistant CmR gene fragment, the structure of which is shown in Figure 6.

Plasmid pTnsPtr comprises the genes tnsA (SEQ ID NO:8), tnsB (SEQ ID NO:9) and tnsC (SEQ ID NO:10) derived from Pseudoalteromonas translucida KMM520, a ColA replicon, a promoter such as an anhydrocycline-inducible promoter 1. Kanamycin resistance gene, commissioned by Nanjing GenScript Biotechnology Co., Ltd. to synthesize, its nucleotide sequence is SEQ ID NO: 22, and its structure is shown in Figure 7.

Plasmid pQCasTnsPtr comprises gene tnsA (SEQ ID NO:8), tnsB (SEQ ID NO:9), tnsC (SEQ ID NO:10), tniQ (SEQ ID NO:11), Cas5/8 ( SEQ ID NO: 12), Cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13), crRNA sequences targeting genomic target sites, ColA replicon, promoters such as anhydrocycline-inducible promoters, card The structure of the namycin resistance gene is shown in FIG. 8 .

Plasmid pQCasTnsVch contains genes Cas5/8, Cas7, Cas6, tniQ, tnsA, tnsB, tnsC and CRISPR array derived from Vibrio cholerae Tn6677, CloDF13 replicon, promoter such as anhydrocycline-inducible promoter, streptomycin resistance gene , whose structure is shown in Figure 9, constructed by Yang Sheng's research group of Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences.

Plasmid pEffectorPtr comprises gene tnsA (SEQ ID NO:8), tnsB (SEQ ID NO:9), tnsC (SEQ ID NO:10), tniQ (SEQ ID NO:11), Cas5/8 ( SEQ ID NO: 12), Cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13), crRNA sequences targeting genomic target sites, LE (SEQ ID NO: 19) and RE (SEQ ID NO: 20) Sequence, target cargo gene, ColA replicon, promoter such as anhydrocycline-inducible promoter, kanamycin resistance gene, the structure of which is shown in FIG. 10 .

Plasmid pEffectorVch contains genes Cas5/8, Cas7, Cas6, tniQ, tnsA, tnsB, tnsC and CRISPR array, LE and RE sequences from Vibrio cholerae Tn6677, target cargo gene, CloDF13 replicon, promoter such as anhydrocycline-inducible The structure of the promoter and streptomycin resistance gene is shown in Figure 11, which was constructed by Yang Sheng's research group of the Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences.

Example 1 Verifying the transposition activity of CRISPR-associated transposases

A CRISPR-associated enzyme from Pseudomonas translucidus KMM520 was demonstrated to have programmable transposition activity by targeting the lacZ and T7 RNA polymerase pro-lacZ of the Escherichia coli BL21Star ^TM (DE3) genome.

The pQCascadePtr-cr3 plasmid targeting genomic lacZ and pre-lacZ of T7 RNA polymerase and the verified primers are listed in Table 1. The suffix "-cr3" in the plasmid name pQCascadePtr-cr3 represents the construction of targeting genome lacZ.

Table 1: Primer sequences

Note: The suffix F in the primer name in the table represents the forward primer, and R represents the reverse primer.

1.1 Enzyme digestion to obtain pQCascadePtr backbone fragment

The pQCascadePtr plasmid was digested with NcoI and BamHI to obtain the backbone fragment of pQCascadePtr.

Enzyme digestion reaction system (50μL)

Enzyme digestion reaction conditions: 37°C, 1h. The digested plasmid fragments were separated by gel electrophoresis and gel recovered.

The restriction endonuclease kit was purchased from Thermofisher Company, and the DNA gel recovery kit was purchased from Shanghai Tolo Harbor Biotechnology Co., Ltd.

1.2 Self-annealing of primers

The primer pair PtrDR-F and PtrDR-R in Table 1 can be annealed and self-assembled to obtain a DR fragment containing a 4bp sticky end, which is complementary to the sticky end of the pQCascadePtr plasmid backbone after NcoI and BamHI digestion.

Annealed self-build system (50μL)

Incubate at 95°C for 5 minutes, lower the temperature by 5-10°C per minute, and incubate at 16°C for 10 minutes, dilute 20 times with ddH ₂ O, and set aside.

1.3 Connection construction plasmid pQCascadePtr-DR

T4 connection system (10μL)

Connect at 16°C for 1h. T4 ligase kit was purchased from TAKARA company.

All the above ligation products were transformed into DH5α competent cells (purchased from Shenzhen Kangti Life Technology Co., Ltd.), and screened on LB solid plates containing streptomycin to obtain plasmid pQCascadePtr-DR. It was verified correct by sequencing with primer 8.

1.4 Enzyme digestion to obtain pQCascadePtr-DR backbone fragment

The pQCascadePtr-DR plasmid was digested with BsaI to obtain the pQCascadePtr-DR backbone fragment.

Enzyme digestion reaction system (50μL)

Enzyme digestion reaction conditions: 37°C, 1h. The digested plasmid fragments were separated by gel electrophoresis and recovered.

The restriction endonuclease kit was purchased from Thermofisher Company, and the DNA gel recovery kit was purchased from Tolo Harbor Company.

1.5 Self-annealing of primers

The primer pair Ptrcr3-F and Ptrcr3-R in Table 1 can be annealed and self-assembled to obtain a DR fragment containing a 4bp sticky end, which is complementary to the sticky end of the pQCascadePtr-DR plasmid backbone after BsaI digestion.

Annealed self-build system (50μL)

Incubate at 95°C for 5 minutes, lower the temperature by 5-10°C per minute, and incubate at 16°C for 10 minutes, dilute 20 times with ddH ₂ O, and set aside.

1.6 Connection construction plasmid pQCascadePtr-cr3

T4 connection system (10μL)

Connect at 16°C for 1h. T4 ligase kit was purchased from TAKARA company.

All the above ligation products were transformed into DH5α competent cells (purchased from Shenzhen Kangti Life Technology Co., Ltd.), and screened on LB solid plates containing streptomycin to obtain plasmid pQCascadePtr-cr3. It was verified correct by sequencing with primer 8.

1.7 Transformation of transposition tool plasmid and induction of transposition

Electrotransform pDonorPtr and pTnsPtr into Escherichia coli BL21Star ^TM (DE3), and select on LB solid plates containing ampicillin and kanamycin at 37°C. The positive clones were selected and made into competent cells for electroporation, and pQCascadePtr-cr3 was electrotransformed into the above-mentioned bacteria, and screened on LB solid plates containing ampicillin, kanamycin and streptomycin at 37°C to obtain cells containing E. coli BL21Star ^™ (DE3) strains of pDonorPtr, pTnsPtr and pQCascadePtr-cr3. Scrape a part of the clones on the above plate, resuspend them in liquid LB medium, reapply on LB solid plates containing anhydrotetracycline, ampicillin, kanamycin and streptomycin at a final concentration of 100ng/ml, anhydrotetracycline Responsible for inducing the expression of transposition-associated enzymes. After culturing at 37°C for 16 hours, a layer of bacterial film may form, which is normal. Scrape a part of the clones on the plate containing 100ng/ml anhydrotetracycline and resuspend in liquid LB medium, adjust the OD ₆₀₀ to about 0.5, dilute 50 times with liquid LB medium, draw 100 μL and apply to the final concentration 1000ng/ml anhydrotetracycline, ampicillin, kanamycin and streptomycin on LB solid plates, cultured at 37°C for 24h.

1.8 Efficiency of colony PCR identification targeting crRNA3

Using the primer pairs crRNA3-F/crRNA3-R and crRNA3-R/T7lacZcr3-R located upstream and downstream of the insertion site in Table 1, verify the efficiency of targeting the two sites of crRNA3 by colony PCR.

Colony PCR reaction system (10μL):

PCRMix was purchased from Novizan.

PCR reaction conditions:

The donor insert on plasmid pDonorPtr includes LE (Left end), RE (Right end) and cargo gene CmR fragment (chloramphenicol resistance gene fragment without promoter), a total of 1433bp, positive band 1601/1759bp, negative Band 168/326bp. According to statistics, all 16 clones had insertions at two sites. The gel electrophoresis picture is shown in Figure 1.

Figure 1 clearly shows the band of the CmR fragment of the cargo gene, which was confirmed to be derived from Pseudoalteromonas translucidus by targeted cloning of the CmR fragment into the lacZ of the Escherichia coli BL21Star ^TM (DE3) genome and the pre-lacZ of T7 RNA polymerase The CRISPR-associated transposase KMM520 has programmable transposition activity.

Example 2 Using array to target 8 different sites in the genome to achieve multi-copy integration

2.1 Construction of plasmid pQCasTnsPtr-array8

Referring to the method of Example 2 in the patent document CN202010083919.7, the crRNA targeting 8 different sites of the Escherichia coli BL21Star ^TM (DE3) genome was combined to form 9 fixed direct repeat sequences and 8 targeting different sites The array sequences arranged at spacer intervals were inserted between the NcoI and BamHI sites of the pQCasTnsPtr plasmid to construct the plasmid pQCasTnsPtr-array8. The array sequence synthesis and the above plasmid construction were completed by Nanjing GenScript Biotechnology Co., Ltd.

2.2 Transformation of transposition tool plasmid and induction of transposition

Electrotransform the plasmids pDonorPtr and pQCasTnsPtr-array8 into Escherichia coli BL21Star ^TM (DE3), and the transformation operation is the same as step 1.7. Plate colony-induced transposition and subculture operations are the same as step 1.7. The cargo gene in the plasmid pDonorPtr is the green fluorescent protein GFP gene (about 1.29kb).

2.3 Colony PCR identification of 8-site insertion in the genome

Forward and reverse primers were designed at the upstream and downstream of the 8 genome sites, namely the primers required for verification. The sequences are shown in Table 2.

Table 2. The sequence of primers required to verify the insertion of 8 sites in the genome

Note: The suffix F in the primer name in the table represents the forward primer, and R represents the reverse primer.

The PCR system and reaction conditions are the same as step 1.8.

Colony PCR and nucleic acid gel electrophoresis were used to verify the insertion of 8 loci in the genome of each clone. As shown in Figure 2, all cargo genes at 8 loci can be inserted in the target colony at one time, and the specific distribution of cargo gene copy numbers is shown in the figure 3. It shows that using the novel CRISPR-associated transposase, the cargo gene insertion efficiency is 100%, which is higher than that of the CRISPR-associated transposase derived from Vibrio cholerae Tn6677.

Example 3 Orthogonal experiments of two CRISPR-related transposases

The orthogonality between a novel CRISPR-associated transposase from Pseudoalteromonas translucida KMM520 and a CRISPR-associated transposase from Vibrio cholerae Tn6677 was investigated.

3.1 Plasmid pQCasTnsPtr-nagman and pQCasTnsVch-cr3 plasmid construction

Combine the crRNA targeting Escherichia coli BL21Star ^TM (DE3) genome nagB, nagE and manX to form an array sequence consisting of 5 fixed direct repeats and 4 spacers targeting different sites, and insert it into the pQCasTnsPtr plasmid Between the NcoI and BamHI sites, the plasmid pQCasTnsPtr-nagman was constructed. The array sequence synthesis and the above plasmid construction were completed by Nanjing GenScript Biotechnology Co., Ltd.

The pQCasTnsVch-cr3 plasmid construction method is the same as steps 1.4, 1.5, and 1.6, and the plasmid pQCasTnsVch comes from our laboratory.

The primers used for plasmid construction and identification are listed in Table 3.

Table 3. Primers used for construction and identification of plasmids pQCasTnsPtr-nagman and pQCasTnsVch-cr3

serial number Primer Sequence (5'→3') 1 Vchcr3-F ATAACTATCCCATTACGGTCAATCCGCCGTTTGTTCCG 2 Vchcr3-R TTCACGGAACAAACGGCGGATTGACCGTAATGGGATAG 3 tetR-seq AGTGAGTATGGTGCCTATCT 4 crRNA3-F CACCACAGATGAAACGCCG 5 crRNA3-R CATCTACACCAACGTGACCTATCC 6 T7lacZcr3-R CACCCATCTCGTAAGACTCATG 7 wxya AACCTCGCATTAATCTTCGC 8 nagER GCAGTAACAGCAACAGAAAGCA 9 wxya TGCAATATGACCGTCGTTACC 10 wxya TGAGACTGATCCCCCCTGACT 11 man XF GAAATGCTGTTAGGCGAGCA 12 manXR TGAATCAGACGGTCGTCGAT 13 manXF2 GATGAAGTGGCTGCGGATAC 14 manXR2 CCTTACGGACTTCCAGCTCCA

3.2 Transformation of transposition tool plasmid and induction of transposition

Electrotransform plasmids pDonorPtr, pQCasTnsPtr-nagman, pDonorVch and pQCasTnsVch-cr3 into Escherichia coli BL21Star ^TM (DE3), and the transformation operation was the same as step 1.7. Plate colony-induced transposition and subculture operations are the same as step 1.7. The cargo gene carried by pDonorPtr is the green fluorescent protein GFP gene (about 1.29kb), and the cargo gene carried by pDonorVch is the terminator sequence (about 0.64kb).

3.3 Identification of Genome Insertion by Colony PCR

Targeting of genomic lacZ and T7 RNA polymerase pre-lacZ with a CRISPR-associated transposase derived from Vibrio cholerae Tn6677 with a cargo gene size of 0.64 kb. The crRNA array of pQCasTnsPtr was designed to target the genomes nagB, nagE and manX, and the cargo gene green fluorescent protein GFP gene (about 1.29kb). After the E. coli transposition experiment, use the upstream and downstream primers of the target site on lacZ for PCR detection, the positive band size should be 1.0kb and 1.17kb, and the negative band size should be 0.17kb and 0.33kb; use nagB, nagE and manX PCR detection of primers upstream and downstream of the upper target site, the positive band size is about 2.47kb, 2.70kb, 2.49kb and 2.36kb, and the negative band size should be 0.43kb, 0.66kb, 0.46kb and 0.33kb. The results are shown in Figure 4. Both the CRISPR-related transposase and pQCasTnsPtr derived from Vibrio cholerae Tn6677 target the corresponding sites, and the corresponding cargo genes are inserted (the cargo gene of pDonorPtr is the green fluorescent protein GFP gene, and the cargo gene carried by pDonorVch is terminator sequence) to obtain 2*4 copies of the strain with an efficiency of about 100%, which was verified by sequencing, see Figure 4.

Figure 4 shows the insertion of the cargo gene GFP carried by Ptr and the "terminator sequence" of the cargo gene carried by Vch at 6 sites in the strain genome, indicating that the two CRISPR-related transposases can be used in the same E. coli, and can Function without interfering with each other, thus providing an option for accelerating the construction of metabolic engineering strains.

Example 4 Verifying the transposition activity of CRISPR-associated transposases in Narophilicus

By targeting the genome wbfF gene of Narophilus ATCC14048, it was confirmed that a CRISPR-associated enzyme derived from Pseudoalteromonas translucidus KMM520 has programmable transposition activity in Narrative Vibrio.

Plasmid pVnQCasTnsPtr comprises gene tnsA (SEQ ID NO:8), tnsB (SEQ ID NO:9), tnsC (SEQ ID NO:10), tniQ (SEQ ID NO:11), Cas5/8 ( SEQ ID NO: 12), Cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13), crRNA sequences targeting genomic target sites, p15A replicon, promoters such as anhydrocycline-inducible promoters, chloride Mycin resistance gene, the plasmid structure is shown in Figure 12.

Plasmid pDonorPtr-GFP contains gene LE (SEQ ID NO:19) and RE (SEQ ID NO:20) sequences derived from Pseudoalteromonas translucida KMM520, pMB1 replicon, ampicillin resistance gene, target cargo gene (Cargo) without promoter The green fluorescent protein gene fragment.

The construction of pVnQCasTnsPtr-wbfF plasmid targeting genome wbfF and the verified primers are listed in Table 4. The suffix "-wbfF" in the plasmid name pVnQCasTnsPtr-wbfF represents the construction of targeted genome wbfF. The pVnQCasTnsPtr-wbfF plasmid construction method is the same as steps 1.4, 1.5, and 1.6.

Table 4: Primer sequences

Numbering Primer Sequence (5'→3') 1 wbfF-F GAAATCTCTCTTTGGTATCTCTATCAGTCTACTCTAAG 2 wxya TTCACTTAGAGTAGACTGATAGAGATACCAAAAGAGAGA 3 wbfF-test-F TCAGCAGAACAACGACACTCAG 4 wbfF-test-R TATTGCCTTGGTTATTCTACCGTGAC 5 tetR-seq AGTGAGTATGGTGCCTATCT

Note: The suffix F in the primer name in the table represents the forward primer, and R represents the reverse primer.

4.1 Transformation of transposition tool plasmid and induction of transposition

Electrotransform pDonorPtr-GFP into sodium vibrio ATCC14048, and select on LBv2 solid plate containing ampicillin at 30°C. Select the positive clones and make them into competent cells for electroporation, then electrotransform pVnQCasTnsPtr-wbfF into the above bacteria, screen on LBv2 solid plates containing ampicillin and chloramphenicol at 30°C, and obtain pDonorPtr-GFP and pVnQCasTnsPtr -wbfF of the Narophilicus ATCC14048 strain. Scraped a part of the clones on the above plate, resuspended in liquid LBv2 medium, and reapplied on LBv2 solid plate containing anhydrotetracycline, ampicillin and chloramphenicol at a final concentration of 100ng/ml. Anhydrotetracycline is responsible for inducing transposition association Enzyme expression. After culturing at 30°C for 12 hours, a layer of bacterial film may form, which is normal. Scrape a part of the clones on the plate containing 100ng/ml anhydrotetracycline and resuspend in liquid LBv2 medium, adjust OD ₆₀₀ to about 0.5, dilute 50 times with liquid LBv2 medium, draw 100μL and spread to the final concentration 1000ng/ml anhydrotetracycline, ampicillin and chloramphenicol on LBv2 solid plate, cultured at 30°C for 12h.

4.2 Efficiency of colony PCR identification targeting wbfF gene

The efficiency of targeting the two sites of wbfF was verified by colony PCR using the primer pair wbfF-test-F/wbfF-test-R located upstream and downstream of the insertion site in Table 4. The colony PCR method is the same as step 1.8.

The donor insert on the plasmid pDonorPtr-GFP includes LE (Left end), RE (Right end) and cargo gene GFP fragment (green fluorescent protein gene fragment without promoter), a total of 720bp, positive band 1220bp, negative band 500bp. According to statistics, all 16 clones had insertions. We demonstrate that a CRISPR-associated transposase derived from Pseudoalteromonas translucidum KMM520 also has programmable transposition activity in Narophilicus.

Example 5 verifies the transposition activity of CRISPR-related transposase in Corynebacterium glutamicum

The plasmid pCgQCasTnsPtr used in the examples comprises genes tnsA (SEQ ID NO:8), tnsB (SEQ ID NO:9), tnsC (SEQ ID NO:10), tniQ (SEQ ID NO:11) derived from Pseudoalteromonastranslucida KMM520 , Cas5/8 (SEQ ID NO: 12), Cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13), the above-mentioned genes are all codon-optimized in Corynebacterium glutamicum, targeting the crRNA at the genomic target site The structure of sequence, ColA replicon, pBL1ts replicon, promoter such as anhydrocycline-inducible promoter, kanamycin resistance gene is shown in FIG. 13 .

Plasmid pCgDonorPtr contains gene LE (SEQ ID NO: 19) and RE (SEQ ID NO: 20) sequences derived from Pseudoalteromonas translucida KMM520, pMB1 replicon, pGA1 replicon, spectinomycin resistance gene, target cargo gene Cargo, for example, no The structure of the chloramphenicol-resistant CmR gene fragment of the promoter is shown in FIG. 14 .

The transposition activity of CRISPR-associated enzymes from Pseudomonas translucidum KMM520 in Corynebacterium glutamicum was confirmed by targeting the crtYf gene of Corynebacterium glutamicum ATCC13032 genome.

The crRNA sequence targeting the genome crtYf gene is constructed as follows:

AGGCAACCATAGGGCAGGAATCAGAAGTACTG.

5.1 Transformation of transposition tool plasmid and induction of transposition

Electrotransformation of pCgDonorPtr and pCgQCasTnsPtr into Corynebacterium glutamicum ATCC13032 was performed at 30°C on a BHIS solid plate containing spectinomycin and kanamycin to obtain Corynebacterium glutamicum ATCC13032 strains containing pCgDonorPtr and pCgQCasTnsPtr. Scraped a part of the clones on the above plate, resuspended in liquid BHIS medium, and reapplied on the BHIS solid plate containing anhydrotetracycline, spectinomycin and kanamycin at a final concentration of 100ng/ml. Anhydrotetracycline is responsible for inducing transformation. Expression of loci-associated enzymes. After culturing at 30°C for 24 hours, a layer of bacterial film may form, which is normal. Scrape a part of the clones on the plate containing 100ng/ml anhydrotetracycline and resuspend in liquid BHIS medium, adjust the OD ₆₀₀ to about 0.5, dilute 50 times with liquid BHIS medium, draw 100μL and apply to the final concentration 1000ng/ml anhydrotetracycline, spectinomycin and kanamycin on the BHIS solid plate, cultured at 30°C for 48h.

5.2 Efficiency of colony PCR identification targeting crtYf

The efficiency of targeting crtYf was verified by colony PCR using the primer pair F-crtYf (TGCTGTGGGAACTTTTCGGT) and R-crtYf (ACTACCACTCCCGAGGTTGA) located upstream and downstream of the insertion site.

The donor insert on plasmid pDonorPtr includes LE (Left end), RE (Right end) and cargo gene CmR fragment (chloramphenicol resistance gene fragment without promoter), a total of 1433bp, positive band 2110bp, negative band 677bp. According to statistics, all 6 clones were inserted successfully. The gel electrophoresis image is shown in Figure 15, confirming the transposition activity of the CRISPR-associated transposase derived from Pseudoalteromonas translucenta KMM520 in Corynebacterium glutamicum.

sequence listing

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210 215 220

Gln Leu Thr Thr Asp Arg Lys Leu Asn Phe Ala Leu Val Glu His Ser

225 230 235 240

Arg Pro Ala Asn Val Gly Asp Leu Ala Ser Ser Val Gly Gly Asn Ile

245 250 255

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

260 265 270

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

275 280 285

Leu Leu Ser Ser Gln Phe Gln Gln Ala Leu Leu Val Leu Val Gly Ile

290 295 300

Lys Gln Phe Asn Thr Leu Arg Gln Lys Arg Leu Ala Arg Val Ala Ala

305 310 315 320

Ile Arg Gln Val Arg Val Ser Leu Gln Leu Trp Leu Asp Asn Ile Leu

325 330 335

Glu Ala Lys Asn Asn Ala Gln Asn Gln Val Tyr Pro Glu Trp Val Arg

340 345 350

His Tyr Leu Asp Gln Ser Ile Thr Asn Cys Ile Ser Gln Phe Ser Asn

355 360 365

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

370 375 380

Tyr His Pro Asn Leu Met Gly Leu Phe Lys Ala Gln Leu Asn Tyr Val

385 390 395 400

Phe Thr His Cys Ala Ala Glu Gln Glu Ile Leu Asn Asp Glu Gln Ile

405 410 415

Val Tyr Val His Cys Gln Asp Met Arg Val Phe Asp Ala Glu Ala Met

420 425 430

Ala Asn Pro Tyr Ile Gln Gly Met Pro Ser Leu Thr Ala Leu Asn Gly

435 440 445

Leu Ala His Asn Phe Glu Arg Lys Leu Lys Asn Phe Ile Asp Pro Ser

450 455 460

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

465 470 475 480

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

485 490 495

Arg Ser His Val Ile Arg Ser Gly Ile Ile Asp Lys Pro Lys Cys Asp

500 505 510

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

515 520 525

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

530 535 540

Phe Ala Gly Gly Thr Met His Pro Pro Ser Leu Tyr Gln Asn Ile Asp

545 550 555 560

Trp Cys His Val His Thr Lys Pro Ser Glu Leu Phe Lys Lys Leu Lys

565 570 575

Ala Lys Ser Ser Asn Gly Ser Trp Leu Tyr Pro Ser Lys Lys Val Val

580 585 590

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

595 600 605

Arg Pro Ala Ala Ile Gly Leu Ala Ala Leu Glu Glu Pro Val Lys Arg

610 615 620

Asp Ala Ala Leu His Glu Tyr His Cys Tyr Ala Glu Pro Val Ile Gly

625 630 635 640

Leu Leu Glu Cys Val Ser Asn Thr Ser Val Lys Tyr Ala Gly Ala Lys

645 650 655

Gln Phe Phe His Asp Ala Phe Trp Val Met Asp Val Gln Lys Glu Ser

660 665 670

Met Leu Met Lys Lys Ser Lys Phe Glu Tyr Glu

675 680

<210> 6

<211> 200

<212> PRT

<213> Pseudoalteromonas translucida KMM520

<400> 6

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

1 5 10 15

Val Ser Leu Leu Ala Gly Arg Cys Ile Gly Ile Leu His Gly Phe Met

20 25 30

Ser Ser Arg Glu Ile Ser Asn Ile Gly Val Cys Phe Pro Lys Trp Asn

35 40 45

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

50 55 60

Gln Leu Thr Asn Leu Ser Gln Gln Ser Tyr Phe Glu Met Met Ala His

65 70 75 80

Asp Lys Leu Phe Gly Leu Ser Lys Ile Leu Glu Val Pro Val Asn Gln

85 90 95

Ser Glu Val Met Phe Val Arg Asn Gln Ser Val Ala Lys Ala Phe Val

100 105 110

Gly Glu Lys Gln Arg Arg Leu Lys Arg Ala Lys Lys Arg Ala Glu Ala

115 120 125

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

130 135 140

Gly His Phe His Ser Ile Pro Val Ser Ser Lys Gly Asn Gly Gln Ser

145 150 155 160

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

165 170 175

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

180 185 190

Val Pro Ser Leu Asn Thr Leu Leu

195 200

<210> 7

<211> 342

<212> PRT

<213> Pseudoalteromonas translucida KMM520

<400> 7

Met Gln Leu Pro Arg His Leu Ser Tyr Thr Arg Ser Leu Ser Pro Ser

1 5 10 15

Lys Ala Val Phe Phe Tyr Lys Thr Pro Glu Ser Asp Phe Glu Pro Leu

20 25 30

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

35 40 45

Ala Tyr Gln Lys Gln Asn Val Ala Lys Asn Leu Ala Pro Gln Asp Leu

50 55 60

Ala Phe Gly Asn Pro Gln Thr Ile Asp Val Cys Tyr Val Pro Pro Thr

65 70 75 80

Val Asn Glu Leu Phe Cys Arg Phe Ser Leu Arg Val Glu Ala Asn Cys

85 90 95

Ile Glu Pro His Val Cys Asp Asp Pro Lys Val Ile Tyr Trp Leu Lys

100 105 110

Arg Phe Phe Glu Thr Tyr Lys Lys His Asn Gly Leu Asn Glu Val Ala

115 120 125

Thr Arg Tyr Ala Lys Asn Ile Leu Met Gly Asn Trp Leu Trp Arg Asn

130 135 140

Arg Gln Ser Pro Asn Val Asp Ile Glu Ile Leu Thr Glu His Ala Ala

145 150 155 160

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

165 170 175

Gln Asn Asn Gln Thr Ala Leu Leu Thr Leu Ser Glu Ser Ile Gln Glu

180 185 190

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

195 200 205

Ile Lys Asn Ala Phe Ser Gln Glu Val His Pro Ser Gln Lys Phe Val

210 215 220

Asp Asn Val Glu Gln Gly Met Ser Ser Lys Gln Leu Ala Tyr Thr Gln

225 230 235 240

Val Gly Asp Lys Lys Ala Ala Ser Leu Asn Ser Gln Lys Val Gly Ala

245 250 255

Ala Ile Gln Thr Ile Asp Asp Trp Tyr Glu Glu Gly Tyr Lys Pro Leu

260 265 270

Arg Thr His Glu Tyr Gly Ala Asp Lys Gln Ile Leu Val Ala His Arg

275 280 285

Thr Pro Lys Ser His Ser Asp Phe Tyr Ser Leu Leu Pro Arg Ile Ala

290 295 300

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

305 310 315 320

Ser Asn Ser Ile His Phe Ile Ala Ala Val Leu Ile Lys Gly Gly Leu

325 330 335

Phe Gln Arg Ser Lys Gly

340

<210> 8

<211> 630

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<400> 8

atgtacagaa gaaaactaaa atactcccgt gtaaaaaatc ttcataaatt tgctagtcaa 60

aaaaataaat ctacttgttt agtcgaatcc tctttagagt ttgatgcgtg tttccatttt 120

gaattttcac caccaatagc cgcatttgaa gcacaacctc taggttacga atatgagttc 180

gataaccgta tttgccgtta cacacctgac tttttactta cccacacaga cggcacgcaa 240

aaatttatag aagtaaaacc gcaaagcaaa attgctgacg aagactttcg tgcacgtttt 300

attgaaaagc aagccatagc taagcaagat ggacgcgact taatactggt tactgataaa 360

caaatccgtg tatacccaac actcaataac ttaaagcttt tgcatcgcta ctctggtttt 420

cagtctttaa cagaattgca agcatcggta ctagaacttg ttaagcagta cggctctatc 480

aaagtgggcc agttaatcag atatttaaaa gtaactgccg gtgagctact tgctacggtg 540

cttcgcttac tatcactagg gcagttattt gccgacttaa ctacaaatga aatatcaata 600

gaaacagcaa tttggtctaa caatgtttaa 630

<210> 9

<211> 1824

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<400> 9

atgtttaata acgatttgtt tgatgatgag tttaaccagc cattaccaaa agctgaaacc 60

aaactacctc aaaattacac taaagactta caagcccttc ctgaaaaaat aaaaacaaca 120

acatttgcta agcttaaata tattcaatgg cttgaggcta atattcaagg tggttggaca 180

caaaaaaatc ttgaaccttt attaaaatta atgcctgatg ttgagggtga aaaaaagcca 240

agttggagaa cagccgcacg atggtatagc gcttacacca atgcggataa aaatattatg 300

gcgctaatac caagccacca aaaaaagggt aataggggagc gcgatacaac cactgataag 360

ttttttgaaa aagcacttga gcgttactta gtaaaagaaa aaccatcagt ggcttcggct 420

tacaagttct ataaagactt agttattatc gaaaacgaca gtgttgttga cagtgtttta 480

aagcctttaa catacaaagc gtttaaaaac agaaagata acttaccgca atacgaagta 540

atgattgctc gttatggtaa gcgccttgct gatattgctt ataataaggt tgaagggcat 600

aaacggccta tccgagtact tgaaaaagtt gaaattgacc atacgccact tgatcttatt 660

ttattagatg atgagctaca tattccacta ggtaggccta cactcaccat gttggtagat 720

gtgtatagcc attgtattgttggctattac tttagcttca gtgagcctag ctatgatgca 780

gtaaggcgag caatgctaaa tgcgatgaaa cctaaaagtg aagtggcaaa actataccct 840

gatacgatta atgagtggaa gtgtgctggc aaaattgaaa cactcgttgt tgataatggc 900

gctgaatttt ggagcaacag ccttgaactt gcttgtgaag aaataggcat taatactcaa 960

tataacccag tcgcaaagcc ttggttaaaa ccatttgtag aacgtatgtt tggaacaata 1020

aatactgagt tattagatcc tgttcccggt aaaacctttt ctaacatttt acaaaagcat 1080

gaatacaatc caaaaaaaga tgcaatcatg cgctttacga cctttatgca gttatttcat 1140

aaatgggtag tagacgttta tcatcaagat gccgacagtc gctttaagta cataccgagt 1200

caactgtggg atcaaggttt taatacgtta ccaccaacaa tgctaagtga tgctgatctt 1260

caacaactag atgttgtgct cagtatttca aatcatcggg tacttcgtaa aggtgggata 1320

cggctagaaa acttaagcta cgacagtact gaactggcca attatagaaa gcaatttagc 1380

cataaagtat ctcaagaagt ttaattaaa ttaaatcccg atgatatttc ttatatat 1440

gtttaccttg ataagctaga gcattacata aaagtgccat gcatagatcc aaacggttac 1500

acccaaaatt taagtttgaa tcagcataaa ataaatatac gcatccaccg cgactttatt 1560

tcggggctcta tcgataatgt aggcttagca aaagcgcgca tgtttatca taacaaaatt 1620

caaaacgagt ttgaagagtt aaaaaatgcg ccaaaacact caaaagtaaa gggtggtaaa 1680

gcgttagcta aacatcaaaa tatcagtagt gactcacaaa agtcaataac gcatagcaaa 1740

cccgtagagg ccaaaaaggt tacacctaaa gagcaaccaa ctgatagctg ggatgatttt 1800

atctcagact tagatggatt ttaa 1824

<210> 10

<211> 1002

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<400> 10

atgctgaccg ataaacaaaa agaaaagctg aatgaatttc gtgatgtatt tattgaatac 60

ccaataataa ccaccatatt taacgacttc gatagattaa gacttggtaa agggctaaca 120

ggtgaaaagc cttgcatgct cttaaatggc gatacaggca caggtaaaac agcactgatc 180

aagcaatata aagaacgaca tttaccgcaa tttattaatg gtgttatgaa ccaccctgta 240

ttggtaagcc gcatacctag taacccgaca ttagaatcta ctttagcaga gcttcttaaa 300

gatttagggc aagtaggcag cacagagcgt aagctacgaa taaacggcac tcgcttaacg 360

acatcattaa taaaatgcct aaaaacatgt ggcacagagc ttataattat tgatgagttc 420

caagagctaa ttgagcacaa ccaaggtaaa aagcgccgcg agattgctaa tcgattaaaa 480

tatattaacg acgaagcggg tgtatcaatt gtattggtag gtatgccgtg ggcagaaaaa 540

atagcagacg agccccagtg gtcatctcgt ttattaataa ggcggcagtt gccttatttt 600

aagttgtcag aaaacccaaa gcattttgta caactaataa ttggtctagc caaccgtatg 660

ccatttgccg aaaagccaaa cttaagtgag caagcaacag tgtttacttt gttctcatta 720

tcaaaaggtt gctttagaac attaaaatac tttttagatg atgccgtact ttatgcatta 780

atggacaacg cgaaaactct cacaaccaag catttagtta aagcatttga ggtactcttt 840

ccggatgttc ctaatttatt taccttgcct gtagcagaaa taacagcaag cgaagtcgag 900

cgctattcac tttataagcc tgaaagctct caagatgaag acccgtttat agcgaccaag 960

tttactgacc ggatgccgat tagtcagttg ttaaggaaat aa 1002

<210> 11

<211> 1176

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<400> 11

atgcattttt tagttcaaac aaaatcttac ccagatgagg cgcttgaaag ctatttgctg 60

aggcttgcaa gggataactc atacaatggc tatagtgagc ttgctgatat tttgtggcaa 120

tggcttgcag agcaagataa tgagcttgaa ggtgcgctgc cgttagcgct gagtaaagtt 180

gatgtttatc atgctaggca agcgagcagc tttagaataa gagcgcttaa gttggttgct 240

caattagcag atgtaaacgc tggtgacatt cttgcacttg cttggaggcg cagtaatttt 300

aaatttggca accttgccgc agtaagtcga aatgaactgg ctattcccct tgagctactt 360

cgtactgata acatacctgt ttgcattaaa tgcttgtctg aatcttccca tattcccttt 420

tattggcatt taaagcccta taaggcgtgt cataagcata agtcacaatt aattacacgt 480

tgtaaggagt gctatgactt aattgattac agagcctctg aggcgttttt agagtgtgtt 540

tgcggttgta aaataaccaa tagtgaacag ttaaacgatg cagactttaa aattgcaatt 600

gcgcttgcaa gtagtaacag ccaaaaaata gtaggggttga tatcgtggtt tgcgaaggtt 660

aagcaacttg atgtaagtga tgcagacttt aactgcgctt ttgttgatta ctttaatact 720

tggcctgaaa gccttaccac tgaattagat ttactcacaa ataatgcgcg actcaagcaa 780

cttaaccctt ttaataaaac taagttcagc tctgtttatg gcgatttaat ccgtgatggt 840

caaatagctg caacaagtaa ccggaaaaac aaagtaattg atgagattat tagttatttt 900

gtcgaattag ttgatagtaa ccctaaagct aaacatccaa atattggtga cttactgctt 960

tgtacttttg atgccgcagt attgttaaac actactacag agcaagttta caggcttcat 1020

caagaagcct ttttaaactg tgcttattca caaaaaaagc acgaacagct cagagctgat 1080

agccatgtat tttattacg ccaagtgatt gaactacaac aagcattcgc agctgaaaag 1140

cctctaacaa aaaaacaatt tatagcgccg tggtaa 1176

<210> 12

<211> 2052

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<400> 12

atgaacttac aagatgcact tgcaattgaa ccactaaaag aaaaaaccac agcacttaga 60

aaattgttcg ttccatacac gtctcatgtc gaggtagatg gctttgaaga actagcgctg 120

actgtgctca ttaatcttgt ttataagcga agtgagattg atgatttaac aagtgcaaga 180

actgctaaaa gtgtactacg cgatgaagtg ttactgagta agtgcattaa cgaagtgaaa 240

tggtttcata ctcataattt aaaatacccc gatatacgag taagccatca acgtttaatt 300

agtgaagttg taagtgaaga tattgcgggc atttgcagcc ggtcattacc tttaagtttt 360

ggctggtcgc acaacagtgc tgaaattaat catgcaaagc tatttttaac ctcgtttaat 420

tggcaaggtg aagtgacttg tttagcaagg ctgttaatta atgaagagcc tgtttggatt 480

aatttaataa gagcatacgg gtttactaaa aaggcggttt tagaaatctc gggtaaaata 540

aaacagcagt tgccagtggc agagttccca ttagaagtaa gctctttttc accacaatta 600

caaatgccat ttcagcaaag ctaccttgtg gttacgcctg tagtaagcca cgcaatgctg 660

gctaaaattc agcaattaac aacagatcgt aagttaaatt ttgctttagt tgagcactca 720

agacctgcca atgttggcga tttagcaagc tcagtaggcg gcaatataag agtgctgcgt 780

tactttccta aaacatattc aaaggctgtt aaccgctcta aagtagccaa taatgatatt 840

gagaaagcat ttaaaattcg tgcgctatta agtagtcaat ttcaacaggc gcttttggtg 900

ttggtaggca ttaaacagtt taatacgtta aggcaaaaac gattagcgcg agtagcggct 960

attaggcaag tacgtgttag cttgcagtta tggcttgata atattcttga agctaaaaat 1020

aacgcgcaaa accaagttta ccctgagtgg gtaaggcatt acttagatca gagtattact 1080

aactgtatta gccaatttag taacgtacta aatgagagcc ttggtaattt aagtaagctc 1140

aaacgctttg cgtatcaccc taatttaatg ggactgttta aagcgcagtt aaactatgta 1200

tttactcact gtgcagctga acaagaaata ttaaatgatg agcagatagt gtatgtacat 1260

tgccaagata tgcgagtgtt tgatgctgag gcaatggcta atccgtatat tcaaggcatg 1320

ccgtcactta ctgctttaaa tgggcttgct cataactttg agcgtaagct aaaaaacttt 1380

atagaccctt caattaagtg tattggcagt gctatttaca ttgaaaacta tcaattacat 1440

acaggtaaac cattacctga gccaagcaag ttaaaacaag ttgcagggcg tagtcatgta 1500

ataagatctg gaattatcga taaaccaaaa tgtgacataa cactcgattt agtatttaga 1560

ctttttgtac caaatactga gctgttagat aagttaaata gtcagcttat aaagcccgca 1620

ctaccgtctt catttgcagg cgggactatg catccacctt cgttattca aaatattgac 1680

tggtgccatg tacataccaa accgagcgag ctgtttaaaa aacttaaagc aaaatcgtca 1740

aatggcagtt ggttatatcc ttcaaaaaaa gtagttaaaa gttttgaaca attaattgat 1800

gcccttaaca gtaactttaa tttaagaccc gctgcaattg gcttggctgc gcttgaagaa 1860

cccgtaaagc gagatgcagc attacatgaa taccattgtt atgcagagcc cgtaattggg 1920

ctgttagagt gtgttagcaa tacatcagta aagtacgcag gggctaagca gttctttcat 1980

gacgcatttt gggttatgga tgttcaaaaa gagtctatgc ttatgaaaaa gtctaagttt 2040

gagtatgaat aa 2052

<210> 13

<211> 603

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<400> 13

ttgaagcgct attattttac cattacttat ttaccccaaa gttgtgatgt aagccttctt 60

gctgggcgtt gtatcggtat tttgcatggg tttatgagct cacgtgaaat aagtaatatt 120

ggtgtgtgct ttcctaaatg gaatgagcaa acaataggta atgaattagc gtttgtatca 180

acaaataaaa agcaattaac caatctatct cagcaaagct attttgagat gatggctcat 240

gacaagttat ttggcttatc aaaaatactt gaagtaccag taaaccaaag cgaagtcatg 300

tttgttcgca accaatcggt agcaaaagca tttgttggcg aaaagcaaag gcgattaaag 360

cgagctaaaa aacgagctga agccagaggc gaagtttaca accctgaata taaatttgag 420

gcaaaggaca taggccattt tcattcaata cccgtatcaa gcaaaggcaa tggtcaaagt 480

tatgttttgc atatacaaaa aaatgaaaat gctgaatcca taaaaaatca gtttaacaat 540

tatggctttg ctacaaatca aatatttcta ggtacggttc cttctttaaa taccctttta 600

taa 603

<210> 14

<211> 1029

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<400> 14

atgcaattac ctcggcactt aagttacacg cgttcgctct cacccagtaa agcggtgttt 60

ttttataaaa caccagagtc tgactttgaa ccgctacaaa tagagcaaaa taaattagtt 120

gggcagaagt cagggtttgg cgatgcgtat caaaagcaaa atgtggctaa aaatttagcg 180

ccacaagatc tcgcgtttgg aaaccctcaa acaattgatg tgtgttatgt acctccaacg 240

gtaaatgagc tattttgtcg tttttcactc agggttgagg ctaattgtat tgagccacat 300

gtatgtgatg accctaaagt tattattgg ttaaaacggt ttttcgaaac ctataaaaaa 360

cacaatggcc ttaatgaagt tgcaacgcgc tatgctaaaa atatactgat gggcaactgg 420

ctttggcgta accgccaatc accaaatgtt gatattgaaa tccttactga gcacgcagcc 480

ccgattgttg ttgaaggtgc acaaaaacta aaatggcaag gcaactggca aaataatcaa 540

acggcattta taacgttgtc agaatctatt caagaagggc taagcaatcc tcaaaattat 600

tgttatttag atataaccgc aaaaattaaa aatgcattta gccaagaggt tcatcctagt 660

caaaagtttg tagataatgt tgaacaaggt atgtcatcta aacaacttgc atatactcaa 720

gtaggcgata aaaaagcagc aagtttgaat tcacaaaaag tagggggctgc tatccaaact 780

attgatgatt ggtatgagga aggttacaaa cctttacgca ctcacgagta tggcgcagat 840

aagcaaatat tagttgcaca cagaacacct aagagccatt cagactttta ttcattactc 900

ccgcgcattg ctttgcatat taaacacatg gaaaagcatg gtttagagca aagtgaacaa 960

tcaaactcaa ttcactttat tgcggcagtg ctgatcaaag gtggcttgtt tcaaaggagt 1020

aaaggttga 1029

<210> 15

<211> 88

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<220>

<221> misc_feature

<222> (29)..(60)

<223> n is a, c, g, or t

<400> 15

gtgaactgcc gagtaggcag ctggaaatnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60

gtgaactgcc gagtaggcag ctgaagtt 88

<210> 16

<211> 88

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<220>

<221> misc_feature

<222> (29)..(60)

<223> n is a, c, g, or t

<400> 16

gtgaactgcc gagtaggcag ctggaaatnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60

gtgaactgcc gagtaggcag ctggaaat 88

<210> 17

<211> 88

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<220>

<221> misc_feature

<222> (29)..(60)

<223> n is a, c, g, or t

<400> 17

gtgaactgcc gagtaggcag ctgaagttnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60

gtgaactgcc gagtaggcag ctgaagtt 88

<210> 18

<211> 88

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<220>

<221> misc_feature

<222> (29)..(60)

<223> n is a, c, g, or t

<400> 18

gtgaactgcc gagtaggcag ctgaagttnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60

gtgaactgcc gagtaggcag ctggaaat 88

<210> 19

<211>632

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<400> 19

ttaattttcc ttaattattt ttaaagttag actgatttag acttggaaaa gcttaatgat 60

tggagagcta aattgactaa tagtattcag tcaagtttaa ttagttttaa gcgatccatg 120

cataactatt tctgtacaat gctatatttt caccaattaa taatttttaa tcaagcctac 180

attatgaaat atactatacc catttgaact cttctatttg taccatgtc ggtagcaaaa 240

acttatgggg ttttgaacgt cacttaaatt gtaagcattt gcgatggagg cgcgtttaga 300

gtcaaccttg attctgatat gctccgaatt tttggtaaga atataagtgt gagagtagct 360

aatgtggata cgcctgagtt aagggaaaaa tgtgaaaatg aaataactcg ttatcatgca 420

aagtgactaa ggttataatc ttccgtttat ggcacatagc agccaactaa acttgacagt 480

attttatgt ggttggcttt ataaaaccag catttggtaa cattatgcca atttttactt 540

caatattatg ccaacataca ctacactaac ggagctgtag cacaataagc tcgtttgtac 600

ttatgccaac ttatacttca aacaacattg gg 632

<210> 20

<211> 113

<212>DNA

<213> Pseudoalteromonas translucida KMM520

<400> 20

ttgggtgttg tttgaagtat aagttgacat atctgtacta aaagatggca taaattggaa 60

gtgtaaggtg gcatagtcta gtatttaacc aaatggttaa atggttgact cac 113

<210> 21

<211> 8138

<212>DNA

<213> artificial sequence ()

<400> 21

cattaattcc taatttttgt tgacactcta tcattgatag agttatttta ccactcccta 60

tcagtgatag agaaaagtga actctagaaa taattttgtt taactttaaa aggagatata 120

ccatgggtga actgccgagt aggcagctgg aaatgagacc tctggtctcg tgaactgccg 180

agtaggcagc tggaaatgga tccgaattcg agctcggcgc gcctgcaggt cgacaagctt 240

gcggccgctc taatctagac atcattaatt cctaattttt gttgacactc tatcattgat 300

agagttatt taccactccc tatcagtgat agagaaaagt gaactctaga aataattttg 360

tttaacttta aaggagatat acatatgttt ttgcaaagac ctaaataacc aacaagaagg 420

agatatacat atgcactttc tggtgcagac caagagctac ccggacgagg cgctggaaag 480

ctatctgctg cgtctggcgc gtgataacag ctacaacggt tatagcgagc tggcggacat 540

cctgtggcag tggctggcgg aacaagataa cgagctggaa ggtgcgctgc cgctggcgct 600

gagcaaggtg gacgtttacc acgcgcgtca ggcgagcagc ttccgtatcc gtgcgctgaa 660

actggtggcg caactggcgg acgttaacgc gggtgatatt ctggcgctgg cgtggcgtcg 720

tagcaacttc aagtttggca acctggcggc ggtgagccgt aacgagctgg cgatcccgct 780

ggaactgctg cgtaccgata acatcccggt ttgcattaaa tgcctgagcg agagcagcca 840

cattccgttt tactggcacc tgaagccgta taaagcgtgc cacaagcaca aaagccagct 900

gatcacccgt tgcaaggagt gctacgacct gattgattat cgtgcgagcg aggcgtttct 960

ggaatgcgtt tgcggttgca aaatcaccaa cagcgaacaa ctgaacgacg cggatttcaa 1020

gatcgcgatt gcgctggcga gcagcaacag ccagaaaatc gtgggcctga ttagctggtt 1080

cgcgaaggtg aaacaactgg acgttagcga cgcggatttc aactgcgcgt ttgttgatta 1140

cttcaacacc tggccggaga gcctgaccac cgaactggac ctgctgacca acaacgcgcg 1200

tctgaagcag ctgaacccgt ttaacaagac caaattcagc agcgtgtacg gtgacctgat 1260

ccgtgatggc caaattgcgg cgaccagcaa ccgtaagaac aaagttatcg acgagatcat 1320

tagctatttt gtggaactgg ttgatagcaa cccgaaggcg aaacacccga aattggtga 1380

cctgctgctg tgcaccttcg atgcggcggt gctgctgaac accaccaccg agcaggttta 1440

ccgtctgcac caagaagcgt ttctgaactg cgcgtatagc cagaagaaac acgaacaact 1500

gcgtgcggat agccacgtgt tctatctgcg tcaggttatc gagctgcagc aagcgtttgc 1560

ggcggaaaaa ccgctgacca agaaacaatt cattgcgccg tggtaactta tgaacctgca 1620

ggatgcgctg gcgattgagc cgctgaagga aaaaaccacc gcgctgcgta agctgttcgt 1680

gccgtacacc agccacgttg aggtggatgg ttttgaggaa ctggcgctga ccgtgctgat 1740

caacctggtt tataagcgta gcgaaattga cgatctgacc agcgcgcgta ccgcgaaaag 1800

cgtgctgcgt gacgaggttc tgctgagcaa gtgcatcaac gaagtgaaat ggttccacac 1860

ccacaacctg aagtacccgg acatccgtgt tagccaccaa cgtctgatta gcgaggtggt 1920

tagcgaagat atcgcgggta tttgcagccg tagcctgccg ctgagctttg gctggagcca 1980

caacagcgcg gagatcaacc acgcgaaact gttcctgacc agctttaact ggcagggtga 2040

agtgacctgc ctggcgcgtc tgctgattaa cgaggaaccg gtttggatca acctgattcg 2100

tgcgtacggt ttcaccaaga aagcggttct ggagatcagc ggcaagatta aacagcaact 2160

gccggtggcg gagttcccgc tggaagttag cagctttagc ccgcagctgc aaatgccgtt 2220

tcagcaaagc tatctggtgg ttaccccggt ggttagccac gcgatgctgg cgaagatcca 2280

gcaactgacc accgaccgta aactgaactt cgcgctggtt gagcacagcc gtccggcgaa 2340

cgttggtgat ctggcgagca gcgtgggtgg caacattcgt gttctgcgtt actttccgaa 2400

gacctatagc aaagcggtga accgtagcaa agttgcgaac aacgatatcg aaaaggcgtt 2460

caaaattcgt gcgctgctga gcagccagtt tcagcaagcg ctgctggtgc tggttggcat 2520

caagcagttc aacaccctgc gtcaaaaacg tctggcgcgt gtggcggcga tccgtcaagt 2580

gcgtgttagc ctgcaactgt ggctggaca cattctggag gcgaagaaca acgcgcagaa 2640

ccaagtgtac ccggaatggg ttcgtcacta tctggatcaa agcatcacca actgcattag 2700

ccagttcagc aacgttctga acgaaagcct gggtaacctg agcaagctga aacgttttgc 2760

gtaccacccg aacctgatgg gcctgttcaa agcgcaactg aactatgtgt ttacccactg 2820

cgcggcggag caggaaatcc tgaacgacga gcaaattgtg tacgttcact gccaggacat 2880

gcgtgttttc gatgcggaag cgatggcgaa cccgtatatc cagggtatgc cgagcctgac 2940

cgcgctgaac ggcctggcgc acaacttcga gcgtaagctg aaaaacttta ttgatccgag 3000

catcaagtgc attggtagcg cgatctacat tgagaactat caactgcaca ccggcaaacc 3060

gctgccggaa ccgagcaagc tgaaacaggt ggcgggtcgt agccacgtta tccgtagcgg 3120

catcattgac aagccgaaat gcgacattac cctggatctg gtgttccgtc tgtttgttcc 3180

gaacaccgaa ctgctggata agctgaacag ccaactgatt aagccggcgc tgccgagcag 3240

ctttgcgggt ggcaccatgc acccgccgag cctgtaccag aacattgact ggtgccacgt 3300

gcacaccaag ccgagcgagc tgtttaagaa actgaaggcg aaaagcagca acggtagctg 3360

gctgtatccg agcaagaaag tggttaaaag cttcgaacag ctgatcgacg cgctgaacag 3420

caactttaac ctgcgtccgg cggcgattgg cctggcggcg ctggaggaac cggtgaagcg 3480

tgatgcggcg ctgcacgagt accactgcta tgcggaaccg gttatcggtc tgctggagtg 3540

cgtgagcaac accagcgtta agtacgcggg cgcgaaacaa ttctttcacg acgcgttctg 3600

ggtgatggat gttcagaagg aaagcatgct gatgaagaaa agcaaatttg agtatgaata 3660

atgcagctgc cgcgtcacct gagctacacc cgtagcctga gcccgagcaa ggcggtgttc 3720

ttttataaaa ccccggagag cgacttcgaa ccgctgcaga tcgagcaaaa caaactggtg 3780

ggtcagaaga gcggttttgg cgatgcgtac cagaagcaaa acgttgcgaa aaacctggcg 3840

ccgcaggacc tggcgtttgg taacccgcaa accattgatg tgtgctatgt tccgccgacc 3900

gtgaacgaac tgttctgccg ttttagcctg cgtgttgagg cgaactgcat cgaaccgcac 3960

gtgtgcgacg atccgaaggt tattactgg ctgaaacgtt tctttgaaac ctataagaaa 4020

cacaacggtc tgaacgaagt ggcgacccgt tacgcgaaga acatcctgat gggcaactgg 4080

ctgtggcgta accgtcagag cccgaacgtt gacatcgaga ttctgaccga acacgcggcg 4140

ccgattgtgg ttgagggtgc gcagaagctg aaatggcaag gcaactggca gaacaaccaa 4200

accgcgctgc tgaccctgag cgagagcatc caggaaggtc tgagcaaccc gcaaaactac 4260

tgctatctgg atatcaccgc gaagattaaa aacgcgttca gccaggaagt gcacccgagc 4320

caaaagtttg tggacaacgt tgaacagggt atgagcagca aacagctggc gtatacccaa 4380

gtgggcgata agaaagcggc gagcctgaac agccagaagg ttggcgcggc gatccaaacc 4440

attgacgatt ggtacgagga aggttataaa ccgctgcgta cccatgagta tggtgcggac 4500

aagcaaatcc tggtggcgca ccgtaccccg aaaagccaca gcgattttta tagcctgctg 4560

ccgcgtatcg cgctgcacat taagcacatg gaaaaacacg gtctggagca gagcgaacaa 4620

agcaacagca tccacttcat tgcggcggtt ctgattaagg gtggcctgtt tcagcgtagc 4680

aaaggatgaa gcgttactat ttcaccatca cctacctgcc gcaaagctgc gatgtgagcc 4740

tgctggcggg tcgttgcatc ggcattctgc acggtttcat gagcagccgt gagatcagca 4800

acattggcgt gtgctttccg aaatggaacg agcagaccat cggtaacgaa ctggcgtttg 4860

ttagcaccaa caagaaacaa ctgaccaacc tgagccagca aagctatttc gagatgatgg 4920

cgcacgacaa gctgtttggc ctgagcaaaa ttctggaagt gccggttaac cagagcgaag 4980

tgatgttcgt tcgtaaccaa agcgtggcga aggcgtttgt tggtgaaaag caacgtcgtc 5040

tgaaacgtgc gaagaaacgt gcggaggcgc gtggcgaagt gtacaacccg gagtataagt 5100

tcgaagcgaa agatatcggt cactttcaca gcattccggt gagcagcaag ggtaacggcc 5160

agagctacgt tctgcacatc caaaagaacg agaacgcgga aagcattaaa aaccagttca 5220

acaactatgg ctttgcgacc aaccaaattt tcctgggcac cgtgccgagc ctgaacaccc 5280

tgctgtaagg taccaccctt aatctgacct aggctgctgc caccgctgag caataactag 5340

cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa cctcaggcat 5400

ttgagaagca cacggtcaca ctgcttccgg tagtcaataa accggtaaac cagcaataga 5460

cataagcggc tattaacga ccctgccctg aaccgacgac cgggtcatcg tggccggatc 5520

ttgcggcccc tcggcttgaa cgaattgtta gacattattt gccgactacc ttggtgatct 5580

cgcctttcac gtagtggaca aattcttcca actgatctgc gcgcgaggcc aagcgatctt 5640

cttcttgtcc aagataagcc tgtctagctt caagtatgac gggctgatac tgggccggca 5700

ggcgctccat tgcccagtcg gcagcgacat ccttcggcgc gattttgccg gttactgcgc 5760

tgtaccaaat gcgggacaac gtaagcacta catttcgctc atcgccagcc cagtcgggcg 5820

gcgagttcca tagcgttaag gtttcattta gcgcctcaaa tagatcctgt tcaggaaccg 5880

gatcaaagag ttcctccgcc gctggaccta ccaaggcaac gctatgttct cttgcttttg 5940

tcagcaagat agccagatca atgtcgatcg tggctggctc gaagatacct gcaagaatgt 6000

cattgcgctg ccattctcca aattgcagtt cgcgcttagc tggataacgc cacggaatga 6060

tgtcgtcgtg cacaacaatg gtgacttcta cagcgcggag aatctcgctc tctccagggg 6120

aagccgaagt ttccaaaagg tcgttgatca aagctcgccg cgttgtttca tcaagcctta 6180

cggtcaccgt aaccagcaaa tcaatatcac tgtgtggctt caggccgcca tccactgcgg 6240

agccgtacaa atgtacggcc agcaacgtcg gttcgagatg gcgctcgatg acgccaacta 6300

cctctgatag ttgagtcgat acttcggcga tcaccgcttc cctcatactc ttcctttttc 6360

aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 6420

tttagaaaaa taaacaaata gctagctcac tcggtcgcta cgctccgggc gtgagactgc 6480

ggcgggcgct gcggacacat acaaagttac ccacagattc cgtggataag caggggacta 6540

acatgtgagg caaaacagca gggccgcgcc ggtggcgttt ttccataggc tccgccctcc 6600

tgccagagtt cacataaaca gacgcttttc cggtgcatct gtgggagccg tgaggctcaa 6660

ccatgaatct gacagtacgg gcgaaacccg acaggactta aagatcccca ccgtttccgg 6720

cgggtcgctc cctcttgcgc tctcctgttc cgaccctgcc gtttaccgga tacctgttcc 6780

gcctttctcc cttacgggaa gtgtggcgct ttctcatagc tcacacactg gtatctcggc 6840

tcggtgtagg tcgttcgctc caagctgggc tgtaagcaag aactccccgt tcagcccgac 6900

tgctgcgcct tatccggtaa ctgttcactt gagtccaacc cggaaaagca cggtaaaacg 6960

ccactggcag cagccattgg taactggggag ttcgcagagg atttgtttag ctaaacacgc 7020

ggttgctctt gaagtgtgcg ccaaagtccg gctacactgg aaggacagat ttggttgctg 7080

tgctctgcga aagccagtta ccacggttaa gcagttcccc aactgactta accttcgatc 7140

aaaccacctc cccaggtggt tttttcgttt acagggcaaa agattacgcg cagaaaaaaa 7200

ggatctcaag aagatccttt gatcttttct actgaaccgc tctagatttc agtgcaattt 7260

atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt 7320

tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg 7380

gtcgagatcc cggtgcctaa tgagtgagct aacttaccgt tgtaaaacga cggccagtga 7440

attcctgatg aatcccctaa tgatttttat caaaatcatt aaggttacca tcacggaaaa 7500

aggttatgct gcttttaaga cccactttca catttaagtt gtttttctaa tccgcatatg 7560

atcaattcaa ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa taattcgata 7620

gcttgtcgta ataatggcgg catactatca gtagtaggtg tttccctttc ttctttagcg 7680

acttgatgct cttgatcttc caatacgcaa cctaaagtaa aatgccccac agcgctgagt 7740

gcatataatg cattctctag tgaaaaacct tgttggcata aaaaggctaa ttgattttcg 7800

agagtttcat actgtttttc tgtaggccgt gtacctaaat gtacttttgc tccatcgcga 7860

tgacttagta aagcacatct aaaactttta gcgttattac gtaaaaaatc ttgccagctt 7920

tccccttcta aagggcaaaa gtgagtatgg tgcctatcta acatctcaat ggctaaggcg 7980

tcgagcaaag cccgcttatt ttttacatgc caatacaatg taggctgctc tacacctagc 8040

ttctgggcga gtttacgggt tgttaaacct tcgattccga cctcattaag cagctctaat 8100

gcgctgttaa tcactttact tttatctaat ctagacat 8138

<210> 22

<211> 6320

<212>DNA

<213> artificial sequence ()

<400> 22

acgatcgtaa aaggatctca agaagatcct ttacggattc ccgacaccat cactctagat 60

ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt 120

gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct 180

ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg 240

ttgcgctgat gaatccccta atgattttta tcaaaatcat taaggttacc atcacggaaa 300

aaggttatgc tgcttttaag accactttc acatttaagt tgtttttcta atccgcatat 360

gatcaattca aggccgaata agaaggctgg ctctgcacct tggtgatcaa ataattcgat 420

agcttgtcgt aataatggcg gcatactatc agtagtaggt gtttcccttt cttctttagc 480

gacttgatgc tcttgatctt ccaatacgca acctaaagta aaatgcccca cagcgctgag 540

tgcatataat gcattctcta gtgaaaaacc ttgttggcat aaaaaggcta attgattttc 600

gagagtttca tactgttttt ctgtaggccg tgtacctaaa tgtacttttg ctccatcgcg 660

atgacttagt aaagcacatc taaaactttt agcgttatta cgtaaaaaat cttgccagct 720

ttccccttct aaagggcaaa agtgagtatg gtgcctatct aacatctcaa tggctaaggc 780

gtcgagcaaa gcccgcttat tttttacatg ccaatacaat gtaggctgct ctacacctag 840

cttctgggcg agtttacggg ttgttaaacc ttcgattccg acctcattaa gcagctctaa 900

tgcgctgtta atcactttac ttttatctaa tctagacatc attaattcct aatttttgtt 960

gacactctat cattgataga gttattttac cactccctat cagtgataga gaaaagtgaa 1020

ctctagaaat aattttgttt aactttaaaa ggagatatac catgtaccgt cgtaagctga 1080

aatatagccg tgttaagaac ctgcacaaat ttgcgagcca gaagaacaaa agcacctgcc 1140

tggtggagag cagcctggaa ttcgacgcgt gcttccactt tgagttcagc ccgccgatcg 1200

cggcgtttga agcgcaaccg ctgggttacg agtatgaatt cgataaccgt atttgccgtt 1260

acaccccgga ctttctgctg accccacaccg atggcaccca gaagttcatc gaggttaagc 1320

cgcaaagcaa aattgcggac gaggattttc gtgcgcgttt catcgaaaag caggcgattg 1380

cgaaacaaga cggtcgtgat ctgatcctgg tgaccgacaa gcagattcgt gtttacccga 1440

ccctgaacaa cctgaaactg ctgcaccgtt atagcggctt tcagagcctg accgagctgc 1500

aagcgagcgt gctggaactg gttaagcagt acggtagcat caaagtgggc caactgattc 1560

gttatctgaa agttaccgcg ggtgaactgc tggcgaccgt gctgcgtctg ctgagcctgg 1620

gccaactgtt cgcggatctg accaccaacg agatcagcat tgaaaccgcg atctggagca 1680

acaatgttta ataacgacct gttcgacgat gagtttaacc agccgctgcc gaaggcggaa 1740

accaaactgc cgcagaacta taccaaggat ctgcaagcgc tgccggagaa gatcaaaacc 1800

accaccttcg cgaagctgaa atacattcaa tggctggagg cgaacatcca gggtggctgg 1860

acccaaaaga acctggaacc gctgctgaaa ctgatgccgg acgttgaggg tgaaaagaaa 1920

ccgagctggc gtaccgcggc gcgttggtat agcgcgtaca ccaacgcgga taagaacatt 1980

atggcgctga tcccgagcca ccagaagaaa ggcaaccgtg aacgtgacac caccaccgat 2040

aagttctttg agaaagcgct ggaacgttac ctggtgaagg agaaaccgag cgttgcgagc 2100

gcgtataagt tctacaaaga cctggtgatc attgaaaacg acagcgtggt tgatagcgtt 2160

ctgaaaccgc tgacctataa ggcgtttaaa aaccgtattg acaacctgcc gcagtatgag 2220

gttatgatcg cgcgttacgg caagcgtctg gcggatattg cgtacaacaa ggtggaaggc 2280

cacaaacgtc cgattcgtgt gctggagaaa gttgaaatcg accacacccc gctggatctg 2340

attctgctgg acgatgagct gcacatcccg ctgggtcgtc cgaccctgac catgctggtt 2400

gacgtttata gccactgcat cgtgggctac tatttcagct ttagcgagcc gagctacgat 2460

gcggttcgtc gtgcgatgct gaacgcgatg aagccgaaaa gcgaagtggc gaaactgtac 2520

ccggcacca ttaacgagtg gaagtgcgcg ggtaaaatcg aaaccctggt ggttgataac 2580

ggcgcggagt tctggagcaa cagcctggaa ctggcgtgcg aggaaatcgg tattaacacc 2640

cagtataacc cggtggcgaa gccgtggctg aaaccgttcg ttgagcgtat gtttggcacc 2700

atcaacaccg aactgctgga cccggttccg ggcaagacct tcagcaacat cctgcaaaaa 2760

cacgaataca acccgaagaa agacgcgatt atgcgtttca ccacctttat gcagctgttt 2820

cacaagtggg tggttgatgt gtatcaccaa gacgcggata gccgtttcaa atacattccg 2880

agccagctgt gggaccaagg ctttaacacc ctgccgccga ccatgctgag cgatgcggat 2940

ctgcagcaac tggatgtggt tctgagcatc agcaaccacc gtgtgctgcg taagggtggc 3000

attcgtctgg agaacctgag ctatgacagc accgaactgg cgaactaccg taagcagttc 3060

agccacaaag tgagccaaga ggttctgatc aaactgaacc cggacgatat tagctacatc 3120

tatgtgtacc tggacaagct ggaacactat attaaagttc cgtgcatcga tccgaacggt 3180

tacacccaga acctgagcct gaaccaacac aagatcaaca ttcgtatcca ccgtgacttt 3240

attagcggta gcatcgataa cgttggcctg gcgaaggcgc gtatgttcat tcacaacaaa 3300

atccagaacg agtttgagga actgaagaac gcgccgaaac acagcaaggt gaaaggtggc 3360

aaggcgctgg cgaaacacca gaacattagc agcgacagcc aaaagagcat cacccacagc 3420

aaaccggtgg aggcgaagaa agttaccccg aaagaacaac cgaccgatag ctgggacgat 3480

ttcatcagcg acctggatgg tttttaatta tgctgaccga caagcagaaa gaaaagctga 3540

acgagttccg tgatgttttt attgaatacc cgatcattac caccatcttc aacgactttg 3600

atcgtctgcg tctgggtaaa ggcctgaccg gcgagaagcc gtgcatgctg ctgaacggtg 3660

acaccggcac cggtaaaacc gcgctgatta aacagtataa ggaacgtcac ctgccgcaat 3720

tcatcaacgg tgttatgaac cacccggtgc tggttagccg tattccgagc aacccgaccc 3780

tggaaagcac cctggcggag ctgctgaaag acctgggtca agtgggcagc accgagcgta 3840

agctgcgtat taacggcacc cgtctgacca ccagcctgat caaatgcctg aagacctgcg 3900

gcaccgaact gatcattatc gatgagtttc aggaactgat tgagcacaac caaggcaaga 3960

aacgtcgtga aattgcgaac cgtctgaaat acatcaacga cgaggcgggt gttagcattg 4020

tgctggttgg catgccgtgg gcggaaaaga tcgcggatga gccgcagtgg agcagccgtc 4080

tgctgatccg tcgtcaactg ccgtatttca aactgagcga gaacccgaag cactttgtgc 4140

agctgattat cggtctggcg aaccgtatgc cgttcgcgga aaaaccgaac ctgagcgagc 4200

aagcgaccgt tttcaccctg tttagcctga gcaaaggctg cttccgtacc ctgaagtact 4260

ttctggacga tgcggtgctg tatgcgctga tggacaacgc gaagaccctg accaccaaac 4320

acctggtgaa ggcgttcgaa gttctgtttc cggatgtgcc gaacctgttt accctgccgg 4380

ttgcggagat caccgcgagc gaggtggaac gttacagcct gtataagccg gaaagcagcc 4440

aggacgagga cccgttcatt gcgaccaaat ttaccgatcg tatgccgatc agccaactgc 4500

tgcgtaagta actcgagccg ctgagcaata actagcataa ccccttgggg cctctaaacg 4560

ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct 4620

tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg 4680

ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt cggggaaatg 4740

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 4800

attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga 4860

ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg 4920

cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca 4980

atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga 5040

gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca 5100

acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt 5160

cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca attacaaaca 5220

ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa 5280

tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac 5340

catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc 5400

agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt 5460

ttcagaaaca actctggcgc atcgggcttc ccatacaatc gtagattgt cgcacctgat 5520

tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt 5580

aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt cctttttcaa 5640

tattattgaa gcatttatca gggttatgt ctcatgagcg gatacatatt tgaatgtatt 5700

tagaaaaata aacaaatagg catgctagcg cagaaacgtc ctagaagatg ccaggaggat 5760

acttagcaga gagacaataa ggccggagcg aagccgtttt tccataggct ccgcccccct 5820

gacgaacatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa 5880

agataccagg cgtttccccc tgatggctcc ctcttgcgct ctcctgttcc cgtcctgcgg 5940

cgtccgtgtt gtggtggagg ctttacccaa atcaccacgt cccgttccgt gtagacagtt 6000

cgctccaagc tgggctgtgt gcaagaaccc cccgttcagc ccgactgctg cgccttatcc 6060

ggtaactatc atcttgagtc caacccggaa agacacgaca aaacgccact ggcagcagcc 6120

attggtaact gagaattagt ggatttagat atcgagagtc ttgaagtggt ggcctaacag 6180

aggctacact gaaaggacag tatttggtat ctgcgctcca ctaaagccag ttaccaggtt 6240

aagcagttcc ccaactgact taaccttcga tcaaaccgcc tccccaggcg gttttttcgt 6300

ttacagagca ggagattacg 6320

Claims (16)

1. A CRISPR-associated transposase comprising a polypeptide selected from the group consisting of: a transposase protein tnsA derived from a bacterium of the genus pseudoalteromonas, a transposase protein tnsB derived from a bacterium of the genus pseudoalteromonas, a transposase protein tnsC derived from a bacterium of the genus pseudoalteromonas, a transposase protein tniQ derived from a bacterium of the genus pseudoalteromonas, a nuclease protein Cas5/8 derived from a bacterium of the genus pseudoalteromonas, a nuclease protein Cas6 derived from a bacterium of the genus pseudoalteromonas, and a nuclease protein Cas7 derived from a bacterium of the genus pseudoalteromonas.

2. The CRISPR-associated transposase of claim 1, wherein the Pseudoalteromonas bacterium is Pseudoalteromonas translucens, preferably wherein the Pseudoalteromonas translucens is Pseudoalteromonas translucens KMM520 (Pseudoalteromonas translucens transfucida KMM 520).

3. The CRISPR-associated transposase of claim 2,

the tnsA is a polypeptide with an amino acid sequence of SEQ ID NO. 1, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology and has the same function with the SEQ ID NO. 1;

the tnSB is a polypeptide with an amino acid sequence of SEQ ID NO. 2, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology and has the same function with the SEQ ID NO. 2;

the tnsC is a polypeptide with an amino acid sequence of SEQ ID NO. 3, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO. 3 and has the same function;

the tniQ is a polypeptide having an amino acid sequence of SEQ ID NO. 4, or a polypeptide having more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology, with SEQ ID NO. 4 and having the same function;

the Cas5/8 is a polypeptide with an amino acid sequence of SEQ ID NO. 5, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology and has the same function with the SEQ ID NO. 5;

the Cas6 is a polypeptide with an amino acid sequence of SEQ ID NO. 6, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with the SEQ ID NO. 6 and has the same function;

the Cas7 is a polypeptide having an amino acid sequence of SEQ ID NO. 7, or a polypeptide having 95% or more homology, preferably 98% or more homology, more preferably 99% or more homology, with SEQ ID NO. 7 and having the same function.

4. A gene encoding the polypeptide as claimed in any one of claims 1 to 3.

5. The gene according to claim 4,

the gene encoding the polypeptide tnsA having the amino acid sequence of SEQ ID No. 1 is the nucleotide sequence of SEQ ID No. 8 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 8;

the gene encoding the polypeptide tnsB having the amino acid sequence of SEQ ID NO. 2 is the nucleotide sequence SEQ ID NO. 9, or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO. 9;

the gene encoding the polypeptide tnsC having the amino acid sequence of SEQ ID No. 3 is the nucleotide sequence of SEQ ID No. 10 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 10;

the gene encoding the polypeptide tniQ having the amino acid sequence of SEQ ID No. 4 is the nucleotide sequence of SEQ ID No. 11, or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 11;

the gene encoding the polypeptide Cas5/8 with the amino acid sequence of SEQ ID No. 5 is the nucleotide sequence SEQ ID No. 12, or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 12;

the gene encoding the polypeptide Cas6 having the amino acid sequence of SEQ ID No. 6 is the nucleotide sequence SEQ ID No. 13 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 13;

the gene encoding the polypeptide Cas7 having the amino acid sequence of SEQ ID NO. 7 is the nucleotide sequence SEQ ID NO. 14 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO. 14.

6. A plasmid, pqqacade or pqqacadeptr, for use in CRISPR transposon systems comprising a gene fragment selected from the group consisting of: a Cas 5/8-encoding gene as claimed in claim 4 or 5; a Cas 6-encoding gene as set forth in claim 4 or 5; a Cas 7-encoding gene as set forth in claim 4 or 5; a tniQ encoding gene as claimed in claim 4 or 5.

7. The plasmid pQCascadePtr of claim 6, wherein the spacer of the crRNA sequence, spacer, is a spacer that targets a single site in the genome or is an array of crRNAs that targets multiple sites in the genome.

8. The plasmid pQCascadePtr of claim 7, wherein the crRNA sequence is a genomic multisite targeted crRNA array, and wherein the repeat region repeat comprises one or more sequences selected from the group consisting of: the nucleotide sequence is repeat1 of SEQ ID NO. 15, the nucleotide sequence is repeat2 of SEQ ID NO. 16, the nucleotide sequence is repeat3 of SEQ ID NO. 17 and the nucleotide sequence is repeat4 of SEQ ID NO. 18, wherein 32N (N32) in the nucleotide sequences of SEQ ID NOs:15-18 are any base A, T, G or C.

9. A helper plasmid pTns or pTnsPtr for CRISPR transposon systems, for use in conjunction with the plasmid pqracadeptr of any of claims 6-8, comprising gene segments selected from the group consisting of: the gene encoding tnsA as claimed in claim 4 or 5; the tnsB-encoding gene as set forth in claim 4 or 5; the tnsC-encoding gene as set forth in claim 4 or 5.

10. A plasmid pqcrastnsptr for use in CRISPR transposon systems, which is the combination of the plasmid pqcratdtr of any one of claims 6 to 8 and the helper plasmid pTnsPtr of claim 9, comprising: the above Cas5/8, cas6, cas7, tniQ, tnSA, tnSB and tnSC genes, crRNA sequence targeting genome target site, colA replicon, promoter, kanamycin resistance gene.

11. A helper plasmid pDonorPtr for CRISPR transposon systems for use with the plasmid pqqascadeptr of any one of claims 6 to 8 and the helper plasmid pTnsPtr of claim 9, comprising gene segments selected from the group consisting of: a Left End (LE) having the nucleotide sequence of SEQ ID NO. 19 or a sequence having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO. 19 and comprising 33bp of the 3' end of SEQ ID NO. 19; the sequence Right End (RE) having the nucleotide sequence of SEQ ID NO:20 or a sequence 27bp more than 80%, preferably more than 85%, more preferably more than 90%, more preferably more than 95% homologous to SEQ ID NO:20 and comprising the 5' end of SEQ ID NO: 20; the Cargo gene of interest (Cargo gene).

12. A plasmid pEffectorPtr for use in a CRISPR transposon system, formed by combining the plasmid pqqascadeptr of any one of claims 6-8, the helper plasmid pTnsPtr of claim 9, and the helper plasmid pDonorPtr of claim 11, comprising: the above Cas5/8, cas6, cas7, tniQ, tnsA, tnsB and tnsC genes, left End (LE) and Right End (RE) sequences, crRNA sequences targeting the genomic target site, colA replicons, promoters, kanamycin resistance genes.

13. A CRISPR transposon system, comprising: the plasmid pqqcacadepptr of any one of claims 6 to 8, the helper plasmid pTnsPtr of claim 9, the helper plasmid pDonorPtr of claim 11; or the plasmid pQCasTnsPtr of claim 10, the helper plasmid pDonorPtr of claim 11; or the plasmid pEffectorPtr of claim 12.

14. The CRISPR transposase system of claim 13, further comprising a Vibrio cholerae (Vibrio cholerae) Tn6677 derived CRISPR transposase-associated plasmid comprising plasmid pQCastnsVch and helper plasmid pDenoroVch,

wherein plasmid pQCastnsVch comprises: cas5/8, cas6, cas7, tniQ, tnSA, tnSB and tnsC genes from vibrio cholerae Tn6677, cloDF13 replicons, a promoter and a streptomycin resistance gene;

the plasmid pDronVch comprises Left End (LE) and Right End (RE) from Vibrio cholerae Tn6677, and a target Cargo gene (Cargo gene).

15. Use of a CRISPR-associated transposase of any of claims 1 to 3, a gene of claim 4 or 5, a plasmid pQCascadePtr of any of claims 6 to 8, a plasmid pTnsPtr of claim 9, a plasmid pqcastnptr of claim 10, a plasmid pDonorPtr of claim 11, a plasmid peffecterptr of claim 12, a CRISPR transposon system of claim 13 or 14 for gene editing.

16. Use of the CRISPR transposon system of claim 13 or 14 in gene editing, for gene editing of gram negative bacteria such as e.

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