CN114231568A - Auxiliary protein for improving DNA repair efficiency and gene editing vector and application thereof - Google Patents

Abstract

The invention provides an auxiliary protein for improving DNA repair efficiency, a gene editing vector and application thereof, and belongs to the technical field of gene editing and gene therapy. The nucleotide sequence of the auxiliary protein for improving the DNA repair efficiency is shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3. The accessory protein fragment provided by the invention is shorter, compared with a RAD51-Cas9n system, the homologous recombination efficiency is higher, and the accessory protein fragment is more suitable for packaging a transgenic virus vector; compared with the Prime editing system, the method can repair the variation of the multi-base and large-fragment DNA, and has stronger repair function.

Description

Auxiliary protein for improving DNA repair efficiency and gene editing vector and application thereof

Technical Field

The invention relates to the technical field of gene editing and gene therapy, in particular to an auxiliary protein for improving DNA repair efficiency, a gene editing vector and application thereof.

Background

At present, human beings lack effective therapeutic means for Genetic Diseases (Genetic Diseases) caused by changes in sequence or structure of Genetic DNA. The current methods of treatment are more focused on preventive measures such as prenatal diagnosis and the like rather than active treatment. Precision medicine (Precision medicine) provides a solution for radical cure of complex genetic diseases, which is a new concept developed along with the progress of gene sequencing and pharmacogenomics, breaks through the bottleneck of traditional medicine in individual diagnosis and special treatment, and covers genetic diseases such as immune inflammation, cancer, heart disease and the like, and other chronic diseases such as diabetes, neurodegenerative diseases and the like. Gene therapy (Gene therapies) and Gene editing (Gene editing) are achieved by introducing a DNA fragment with a correct sequence into cells carrying mutations of a patient to replace a harmful fragment or introducing protein molecules, activating a cell stress-resistant path, closing a pathogenic Gene action path, assisting correct DNA replacement and carrying out fixed-point correction on bases so as to radically treat genetic diseases. Gene editing techniques have been widely used in the field of biological treatment of disease, and have been well evaluated clinically in the field of immunotherapy for some genetic diseases and rare diseases, such as thalassemia, Duchenne muscular dystrophy, and tumors.

Gene editing (Gene editing) refers to the insertion, deletion, modification or substitution of a DNA fragment at a specific position in the genome of a living body. The CRISPR gene editing system was successively elucidated by the teams jennek, m., chrysnski, k., Fonfara, i., Hauer, m., Doudna, j.a., and charpienter, e. (2012). a programmable dual-RNA-guided dna end in adaptive bacterial immunity 337, 816-821), in 2012, by jenner Doudna and emmuelle charpienter, etc., and it only needs 1 sgRNA (single-guided RNA) and Cas (CRISPR-assisted) nuclease to achieve efficient site-directed cleavage, which has become the mainstream of gene engineering technology due to its simple and efficient characteristics. Cas9 is a representative nuclease among others. Cas9 nuclease has HNH and RuvC nuclease domains, is guided by sgRNA to specific genome positions, recognizes PAM (Protospace AdjacentMotif) to be stable, and further forms Double-Strand Break (DSB) at 3bp upstream of 5' of PAM. However, this system has the disadvantage that off-target effects, which can currently be improved by bioinformatically optimizing the design of sgrnas, are present.

Under the cutting action of enzymes such as Cas9, the genome of the cell is induced to generate DSB and spontaneous repair. The spontaneous DNA repair mechanism of cells is various, including Non-homologous End Joining (NHEJ), homologous Recombination (HDR), selective Non-homologous End Joining (altEJ), and single-strand annealing (SSA). In the NHEJ pathway the protein module pulls the fragmented double-stranded DNA together spatially and then is joined by DNA ligase, a process that does not require a template but often introduces random insertions or deletions (indels). HDR-dependent DNA damage repair mechanisms are also one of the DSB-induced mechanisms of spontaneous gene repair. The whole process of HDR requires the involvement of DNA damage stress proteins, including Brac2, BARD1, Rad51, etc. The genome of the cell can carry out accurate repair on the gene segment cut by the Cas9 through HDR according to a template provided by an endogenous sister chromosome or an exogenous gene segment.

The basic strategy of gene therapy is to use a viral (e.g. adenovirus AAV) vector to carry Cas9 protein, sgRNA and a donor DNA template to perform gene editing and repair on mutant DNA of a body cell genome. The HDR mode of DNA repair is the desired mode of gene repair during CRISPR/Cas9 gene editing, but it is a relatively low proportion, typically less than 1%, in cell-spontaneous gene repair processes. While NHEJ-dependent DNA repair accounts for a substantial proportion of this process. Therefore, the important objective of the scientific community is to improve the DNA repair proportion depending on an HDR mode in the CRISPR/Cas9 gene editing process and better realize accurate gene editing and treatment.

Therefore, gene repair by homologous recombination in gene editing has become one of the important strategies for gene therapy. In order to achieve better targeted and precise gene repair, there are currently several approaches:

(1) single base editing. CBE (cysteine Base editor) and ABE (formed by integrating inactivated Cas9 and tRNA adenosine deaminase) have been developed to perform catalytic conversion of chemical groups of cytosine of deoxynucleotides in specific regions of a genome, so as to realize single Base editing. At present, the single base editing technology is widely confirmed, but the risk of RNA mutation caused by off-target still exists, and researchers should pay attention to side effects in aspects such as off-target of transcriptome and the like.

(2) A Prime Editing (Prime Editing) system is used. Cas9n protein and engineered reverse transcriptase are integrated together, and a pegRNA (primary editing gRNA) which can specifically target and encode a target sequence is combined, so that new genetic information is directly written in a specific site of a human cell genome. And then by improving the pilot editing system, indel byproducts are reduced by transiently expressing an MMR (DNA Mismatch repair) inhibitor MLH1dn, and meanwhile, the editing efficiency is remarkably improved by modifying the prime editor, so that a PEmax framework compatible with a PE4 system, a PE5 system and an epegRNA system is established.

(3) The efficiency of embryonic gene Knock-In (KI) was significantly improved by activating the IHR (Interhololog replay) pathway using the Rad51 protein to assist In embryonic gene editing. Studies have shown that fusion expression of hRad51(S208E-a209D) and Cas9n results in higher integration efficiency, lower deletion and off-target rates in HEK293T cells.

(4) The aim of improving the insertion efficiency of the target segment can be achieved by inhibiting other DSB repair pathways such as NHEJ and the like. For example, the use of small molecule drugs NU7441 and KU-0060648 to inhibit DNA-PK, a key member of the NHEJ pathway, in a variety of cells has been investigated to increase the proportion of HDR.

The current sequence length of various precise gene editors (ABE/CBE, Prime editing, etc.) is too long to exceed the capacity limit of the common transgenic vectors. The capacity of adenovirus vector (AAV) commonly used in gene therapy is only 4.7kb at present, the basic framework in the vector is about 1200bp (including 290bp for two ITRs, 750bp for hCMV and 150bp for polyA), and the capacity of the remaining vector is only about 3 kb. The capacity of a Lentivirus vector (Lentivirus) can be about 6-6.5 kb, hCMV and PolyA are removed, and generally, an insert fragment of less than 5kb is not obtained. And the larger the insert, the significantly lower the lentiviral packaging titer. Whereas the Cas9 gene itself is 4.1kb in length. This results in that binary and even ternary vector systems often have to be used when designing gene editor vector systems, which in turn leads to a significant reduction in the efficiency of virus packaging.

Disclosure of Invention

The invention aims to provide a method for screening shorter auxiliary protein, assisting a DNA repair process induced by gene editing and improving the accurate repair ratio of a gene in an HDR (homologous recombination) mode.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an accessory protein for improving DNA repair efficiency, wherein the nucleotide sequence of the accessory protein is shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

The invention also provides an expression plasmid comprising the helper protein.

Preferably, the nucleotide sequence of the expression plasmid is shown as SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6.

The invention also provides a gene editing vector for improving the DNA repair efficiency, which comprises the auxiliary protein, the DNA editing protein and a delivery vector.

Preferably, the DNA-editing proteins include Cas9 protein, CasX protein, Cas Φ protein, Cas12 protein, Cas14 protein.

Preferably, the helper protein is fused to the carbon terminus or nitrogen terminus of the DNA editing protein.

Preferably, the delivery vector includes a viroid vector Lentivirus, an adenoviral vector AAV, a retroviral vector pMSCV, a pCMV cloning vector, and an integration-defective viroid vector.

Preferably, the integration-defective viroid vector system comprises psPAX2-D64V-NC-MS2(Addgene, #122944), pCMV-BRID-Cas9-6XMS2 (plasmid map as shown in FIG. 14 and plasmid sequence as shown in SEQ ID NO: 14).

The invention also provides application of the auxiliary protein, the expression plasmid and the gene editing vector in preparation of products for improving DNA repair efficiency.

Preferably, the DNA repair is DNA repair by homologous recombination.

Preferably, the product for improving the efficiency of DNA repair comprises a drug or an agent for improving the efficiency of DNA repair.

The accessory protein fragment provided by the invention is shorter, compared with a RAD51-Cas9n system, the homologous recombination efficiency is higher, and the accessory protein fragment is more suitable for packaging a transgenic virus vector; compared with the Prime editing system, the method can repair the variation of the multi-base and large-fragment DNA, and has stronger repair function.

Drawings

FIG. 1 is a plasmid map of pcDNA3.1-NLS-BARD1 plasmid;

FIG. 2 is a plasmid map of pcDNA3-BRID plasmid;

FIG. 3 is a plasmid map of pcDNA3-ARD plasmid;

FIG. 4 is a plasmid map of pcDNA3-BRID-ARD plasmid;

FIG. 5 shows fluorescence intensity of 293T cells with GFP + under fluorescent microscope in example 2;

FIG. 6 shows the proportion of 293T cells analyzing GFP + by FACS in group 1 of example 2;

FIG. 7 shows the proportion of 293T cells analyzing GFP + by FACS in group 2 of example 2;

FIG. 8 shows the proportion of 293T cells analyzing GFP + by FACS in group 3 of example 2;

FIG. 9 shows the proportion of 293T cells analyzing GFP + by FACS in group 4 of example 2;

FIG. 10 shows the proportion of 293T cells analyzing GFP + by FACS in group 5 of example 2;

FIG. 11 is a histogram of the proportion of 293T cells analyzed for GFP + by FACS in groups 1-5 of example 2;

FIG. 12 is an electrophoretogram of accessory protein BRID;

FIG. 13 is an electrophoretogram of the BRID-Cas9 fragment;

FIG. 14 is a plasmid map of integration-defective viroid-like vector pCMV-BRID-Cas9-6XMS 2.

Detailed Description

Example 1

Expression plasmids for the accessory proteins BRID, ARD and BRID-ARD were constructed separately.

The expression plasmids pcDNA3-BRID, pcDNA3-ARD and pcDNA3BRID-ARD are constructed by using the fragments of auxiliary proteins BRID, ARD and BRID-ARD as inserted DNA and pcDNA3.1-NLS-BARD1 plasmid as vector skeleton. Wherein, the nucleotide sequences of the accessory proteins BRID, ARD and BRID-ARD are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. The plasmid map of pcDNA3.1-NLS-BARD1 plasmid is shown in FIG. 1, and the nucleotide sequence is shown in SEQ ID NO: 7.

The construction process is as follows: the BRID, ARD and BRID-ARD helper proteins were separately removed, digested simultaneously with BamH1+ Xho1 endonuclease (NEB) at 37 ℃ for 2h, and DNA products (as insert DNA) were separately recovered using a kit (Novozan, cat # DC 301-01); the pcDNA3.1-NLS-BARD1 plasmid was also digested simultaneously with BamH1+ Xho1 endonuclease (NEB Co.) at 37 ℃ for 2h, and DNA products (as vector backbone DNA) were recovered separately using a kit (Novozam, cat # DC 301-01); ligation was then completed with T4 ligase following the following ligation system (Table 1) at 25 ℃ for more than 5 min:

TABLE 1 construction of the ligation System of three expression plasmids

Composition of matter	Dosage of
		5×T4 Ligase Buffer	2μl
Insert DNA	90fmol
		Vector backbone DNA	30fmol
T4 Ligase	1μl
		ddH2O	Make up system to 10. mu.l

After the connection is finished, the three connection products are respectively transformed into DH5 competent bacteria (Scopheraceae, cat number: TSC-C14), the colonies are selected on an LB plate, the colonies are obtained by sequencing identification, and expression plasmids pcDNA3-BRID, pcDNA3-ARD and pcDNA3-BRID-ARD are obtained by sequencing identification. The plasmid map of the expression plasmid pcDNA3-BRID is shown in FIG. 2, and the nucleotide sequence is shown in SEQ ID NO. 4; the plasmid map of the expression plasmid pcDNA3-ARD is shown in FIG. 3, and the nucleotide sequence is shown in SEQ ID NO. 5; the plasmid map of expression plasmid pcDNA3-BRID-ARD is shown in FIG. 4, and the nucleotide sequence is shown in SEQ ID NO. 6.

Example 2

The fluorescent reporter plasmid pDR-GFP induces the DNA repair process of the NHEJ pathway or the HDR pathway of the cell autonomy under the action of CRISPR/Cas 9. Only HDR-dependent repair modalities, cells normally fluoresce green, while GFP fluorescence is weaker or no fluorescence if NHEJ-mediated DNA ligation is repaired.

This example utilizes the fluorescent reporter plasmid pDR-GFP (purchased from Addgene, #26475) as a reporter plasmid for HDR pathway-dependent DNA repair efficiency. The expression plasmids for the three helper proteins constructed in example 1 were tested for DNA repair efficiency for the HDR pathway.

SceGFPgRNA1 is designed aiming at a DNA sequence near an I-SceI enzyme cutting site on a fluorescent reporter plasmid pDR-GFP, and is cloned into a Lenti-sgRNA-CRISPR-v2 vector (purchased from Addge, #52961) through a BsmB1 site to obtain a Lenti-SceGFPgRNA1-CRISPR gene editing vector. The cloning method is as follows:

(1) the lentiviral vector Lenti-sgRNA-CRISPR-v2 was digested with BsmB1 restriction enzyme (NEB) for 2h at 37 ℃. The DNA fragment was recovered using a kit (Novozan, cat # DC 301-01).

(2) The following 2 primer DNA fragments were mixed, denatured at 95 ℃ for 5 minutes, and cooled at 20 ℃ for 20 minutes at room temperature.

SceGFPsgRNA1-for: aaacaagttcagcgtgtccggcta (shown as SEQ ID NO: 8)

SceGFPsgRNA1-rev: aaaatagccggacacgctgaactt (shown in SEQ ID NO: 9).

(3) The purified Lenti-sgRNA-CRISPR-v2 enzyme-cleaved vector was mixed with the denatured 2 primers, and then reacted with 1. mu.l of T4 ligase (Invitrogen) at 25 ℃ for 5min or more, as shown in Table 2:

table 2 construction of a ligation System of Lenti-SceGFPgRNA1-CRISPR vector

Composition of matter	Dosage of
		5×T4 Ligase Buffer	2μl
2 primers	90fmol
		Purified Lenti-sgRNA-CRISPR (v2) enzyme cutting vector	30fmol
T4 Ligase	1μl
		ddH2O	Make up system to 10. mu.l

(4) The ligation product is transformed into Stbl3 escherichia coli competent strain (Strongco, cat # TSC06), and the strain is selected and cloned on LB culture medium, after overnight culture, DNA is extracted, and the clone inserted into sgRNA1 is identified by a sequencing method, and is named as Lenti-SceGFPgRNA1-CRISPR gene editing vector.

The fluorescent reporter plasmids pDR-GFP and Lenti-SceGFPgRNA1-CRISPR gene editing vectors are transfected into 293T cells, then are respectively transferred into expression plasmids pcDNA3-BRID, pcDNA3-ARD and pcDNA3-BRID-ARD, and the influence of the three expression plasmids on HDR pathway DNA repair efficiency is determined. And 293T cells transfected with the fluorescent reporter plasmid pDR-GFP alone are used as blanks, and 293T cells transfected with the fluorescent reporter plasmid pDR-GFP and the Lenti-SceGFPgRNA1-CRISPR gene editing vector are used as controls.

By 6.25X 10⁴Density of cells/well 293T cells were seeded in 24-well plates in DMEM high-glucose medium + 10% fetal bovine serum FBS at 37And growing the film on the wall at the temperature of 24 hours. The transfection premix system was configured as in table 3, with 3 well replicates for each group. The mixed system was prepared by mixing DNA with Lipo8000 liposome (Biyuntian Co.) and standing at room temperature for 10min or more. The corresponding transfection system was added to each well, the 24-well plate was gently tilted and rotated to mix well the well systems, and then the 24-well plate was placed in a cell incubator, and culture was continued with DMEM high-sugar medium + 10% fetal bovine serum FBS at 37 ℃.

TABLE 3 transfection premix System

TABLE 4 composition Table of culture Medium

Composition (per 50ml)	Brand	Dosage of
			DMEM high-sugar medium	HAKATA	45ml
Fetal Bovine Serum (FBS)	Gibco	5ml
			Penicillin-streptomycin solution (100X)	Biyuntian (a Chinese character)	0.5ml

The cells were cultured for 48h, and the fluorescence intensity was observed under a fluorescence microscope. The cells in each well were then replaced with complete medium containing puromycin at a working concentration of 2 μ g/mL, and a short period of drug stimulation was performed to enhance plasmid gene expression and amplify the fluorescence signal intensity without loss of cells. After 24h incubation with puromycin stimulation, the fluorescence intensity was observed under a fluorescence microscope and recorded as figure 5. As can be seen from fig. 5, the fluorescence intensities of group 3, group 4 and group 5 were significantly higher than those of the control group 2. Therefore, the three accessory proteins BRID, ARD and BRID-ARD provided by the invention can effectively improve the HDR pathway DNA repair efficiency.

After photographing, 293T cells were digested and transferred to a flow analysis tube, washed 2 times with DPBS, and then analyzed for the proportion of GFP + fluorescent cells to the total number of cells using a flow analyzer, and quantitative statistics for the HDR-dependent DNA repair ratio were performed, with the analysis results shown in fig. 6 to 11. As can be seen from FIGS. 6 to 11, the three accessory proteins BRID, ARD and BRID-ARD provided by the present invention can effectively improve the DNA repair efficiency to 12% to 15%. However, the BRID fragment is the smallest and only has 96 amino acids, so that the fragment is suitable for being fused with Cas9 protein to be used as a new gene editor.

In the embodiment, 18bp I-sce1 endonuclease is inserted into a GFP gene fragment of a pDR-GFP report vector, and the gene editing vector provided by the invention can completely cut off an 18bp segment by taking 100bp donor homologous arm DNA at the 2 side of an I-sce1 site as an auxiliary, and realize the perfect repair of the fragment. This demonstrates that the gene editing system provided by the present invention can repair multi-base DNA variations. Similarly, under the guidance of the homology arm, the replacement and repair of large-fragment DNA can be realized in an HDR-dependent DNA repair mode.

Example 3

Construction of Gene editing vectors comprising BRID, ARD or BRID-ARD

Amplification of Cas9 fragment

The Cas9 fragment was amplified using the Lenti-sgRNA-CRISPR-v2 plasmid (purchased from Addgene, #52961) as a vector template.

The primer sequence is as follows:

BARD1-Cas9-5-for2：tataggcagtgggctgtcttcagaaatggacaagaagtacagcatcggc(SEQ ID NO:10)；

BARD1-Cas9-5-rev2：taacgcgttcacttatcgtcatcgtctttgtaatcgtcgcctcccagctgagacag(SEQ ID NO:11)。

amplification was performed using the high fidelity PCR kit (Novozam, cat # P505-d1), the amplification system is shown in Table 5, and the amplification procedure is shown in Table 6:

TABLE 5 amplification System for amplification of Cas9 fragment

Composition of matter	Dosage of
		2×Phanta Max Buffer	25μl
dNTP(10mMeach)	1μl
		Primer F (Primer Cas9-5-for2)	2μl
Primer R (Primer Cas9-5-rev2)	2μl
		DNA template(plasmid，Lenti-sgRNA-CRISPR-v2)	1ng
Phanta Max Super-Fidelity DNA Polymerase (high Fidelity DNA Polymerase)	1μl
		ddH2O	Make up system to 50. mu.l

Table 6 amplification procedure for amplifying Cas9 fragment

The PCR product was purified using the kit (Novozan, cat # DC301-01) to obtain a Cas9 fragment of 4.1kb in size.

Second, connecting Cas9 fragment and accessory protein BRID

The electrophoresis chart of the accessory protein BRID is shown in FIG. 12, and the size is 288 bp.

The BRID-Cas9 fragment was amplified using a high fidelity PCR kit (Novozam, cat # P505-d1) using the purified Cas9 fragment and accessory protein BRID fragment as templates.

The primer sequence is as follows:

BARD1-Cas9-5-For1：gttctagaatgccaaagaagaagcggaag(SEQ ID NO:12)

BARD1-Cas9-5-rev2：gccgatgctgtacttcttgtccatttctgaagacagcccactgcctata(SEQ ID NO:13)。

the amplification system is shown in Table 7, and the amplification reaction procedure is identical to that shown in Table 6.

TABLE 7 amplification System for BRID-Cas9 fragments

Composition of matter	Dosage of
		2×Phanta Max Buffer	25μl
dNTP(10mM each)	1μl
		Primer F (Primer Cas9-5-for1)	2μl
Primer R (Primer Cas9-5-rev1)	2μl
		DNA template (plasmid, BRID, Cas9 fragment)	1ng
Phanta Max Super-Fidelity DNA Polymerase (high Fidelity DNA Polymerase)	1μl
		ddH2O	Make up system to 50. mu.l

The PCR product was purified using the kit (Novozan, cat # DC301-01) to obtain a BRID-Cas9 fragment of 4.4kb in size, and the electrophoretogram is shown in FIG. 13.

Thirdly, constructing a Lenti-U6-sgRNA-EF1a-BRID-Cas9 gene editing vector

The BRID-Cas9 fragment amplified and purified in step two and the Lenti-sgRNA-CRISPRv2 plasmid were digested simultaneously with Xba1+ Mlu1 endonuclease (NEB Co.) at 37 ℃ for2 hours, and the two DNA fragments were purified separately with a kit (Novozam, cat # DC 301-01). Ligation was performed by using T4 ligase (Invitrogen) according to the following ligation scheme (Table 8) and the ligation reaction was carried out at 25 ℃ for 5min or more to obtain a ligated product.

TABLE 8 ligation System of Lenti-U6-sgRNA-EF1a-BRID-Cas9

Composition of matter	Dosage of
		5×T4 Ligase Buffer	2μl
BRID-Cas9 fragment	90fmol
		Lenti-sgRNA-CRISPRv2 plasmid	30fmol
T4 Ligase	1μl
		ddH2O	Make up system to 10. mu.l

The ligation product is transformed into DH5 competent bacteria (Prokinensis, Catharaceae, cat # TSC-C14), selected and cloned on an LB plate, and sequenced and identified to obtain a Lenti-U6-sgRNA-EF1a-BRID-Cas9 gene editing vector system.

In this gene editing vector system, sgRNA is transcribed by the U6 promoter, and the BRID-Cas9 protein is expressed by EF1 a. The BRID-Cas9 protein and sgRNA can be simultaneously expressed in cells, and the cleavage of target genes and the accurate repair of an HDR mode can be simultaneously realized. The DNA repair efficiency of the HDR mode of the gene editing vector system is about 16%, compared with the situation that the HDR efficiency of the homologous recombination in the traditional method is only 3%, the method can greatly improve the gene editing efficiency for accurate treatment of disease genes, and provides a new method and thought for gene and cell treatment.

According to the construction method, gene editing vectors Lenti-U6-sgRNA-EF1a-ARD-Cas9 and Lenti-U6-sgRNA-EF1a-BRID-ARD-Cas9 comprising accessory protein ARD and accessory protein BRID-ARD can also be obtained.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Ankelai (Chongqing) biomedical science and technology Co., Ltd

<120> auxiliary protein for improving DNA repair efficiency, gene editing vector and application thereof

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 288

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

atgccggata atcggcagcc gaggaaccgg cagccgagga tccgcaaacc taggaaaagt 60

ttgtttaatg atgcaggaaa caagaagaat tcaattaaaa tgtggtttag ccctcgaagt 120

aagaaagtca gatatgttgt gagtaaagct tcagtgcaaa cccagcctgc aataaaaaaa 180

gatccagaga agaatgaatc atcctcagct agccactgct cagtaatgaa cactgggcag 240

cgtagggatg gacctcttgt acttataggc agtgggctgt cttcagaa 288

<210> 2

<211> 534

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 2

atgccggata atcggcagcc gaggaaccgg cagccgagga tccgccccaa tatggctgtg 60

aaaagaaatc atagaggaga gactttgctc catattgctt ctattaaggg cgacatacct 120

tctgttgaat accttttaca aaatggaagt gatccaaatg ttaaagacca tgctggatgg 180

acaccattgc atgaagcttg caatcatggg cacctgaagg tagtggaatt attgctccag 240

cataaggcat tggtgaacac caccgggtat caaaatgact caccacttca cgatgcagcc 300

aagaatgggc atgtggatat agtcaagctg ttactttcct atggagcctc cagaaatgct 360

gttaatatat ttggtctgcg gcctgtcgat tatacagatg atgaaagtat gaaatcgcta 420

ttgctgctac cagagaagaa tgaatcatcc tcagctagcc actgctcagt aatgaacact 480

gggcagcgta gggatggacc tcttgtactt ataggcagtg ggctgtcttc agaa 534

<210> 3

<211> 684

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 3

atgccggata atcggcagcc gaggaaccgg cagccgagga tccgcaaacc taggaaaagt 60

ttgtttaatg atgcaggaaa caagaagaat tcaattaaaa tgtggtttag ccctcgaagt 120

aagaaagtca gatatgttgt gagtaaagct tcagtgcaaa cccagcctgc aataaaaaaa 180

gatatgaagc tgttgcccaa tatggctgtg aaaagaaatc atagaggaga gactttgctc 240

catattgctt ctattaaggg cgacatacct tctgttgaat accttttaca aaatggaagt 300

gatccaaatg ttaaagacca tgctggatgg acaccattgc atgaagcttg caatcatggg 360

cacctgaagg tagtggaatt attgctccag cataaggcat tggtgaacac caccgggtat 420

caaaatgact caccacttca cgatgcagcc aagaatgggc atgtggatat agtcaagctg 480

ttactttcct atggagcctc cagaaatgct gttaatatat ttggtctgcg gcctgtcgat 540

tatacagatg atgaaagtat gaaatcgcta ttgctgctac cagagaagaa tgaatcatcc 600

tcagctagcc actgctcagt aatgaacact gggcagcgta gggatggacc tcttgtactt 660

ataggcagtg ggctgtcttc agaa 684

<210> 4

<211> 5712

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 4

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta cccaccatgc caaagaagaa gcggaaggtc agtatgccgg 960

ataatcggca gccgaggaac cggcagccga ggatccgcaa acctaggaaa agtttgttta 1020

atgatgcagg aaacaagaag aattcaatta aaatgtggtt tagccctcga agtaagaaag 1080

tcagatatgt tgtgagtaaa gcttcagtgc aaacccagcc tgcaataaaa aaagatccag 1140

agaagaatga atcatcctca gctagccact gctcagtaat gaacactggg cagcgtaggg 1200

atggacctct tgtacttata ggcagtgggc tgtcttcaga agattacaag gatgacgacg 1260

ataagtgact cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt 1320

ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 1380

ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 1440

gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 1500

atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 1560

ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 1620

tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 1680

tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 1740

tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 1800

gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 1860

agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 1920

cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 1980

agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg 2040

tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 2100

agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 2160

tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc 2220

gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 2280

cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 2340

aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac 2400

aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc 2460

ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc 2520

cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc 2580

cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg 2640

cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt 2700

gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc 2760

catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga 2820

ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga 2880

tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct 2940

caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc 3000

gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt 3060

ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg 3120

cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat 3180

cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc 3240

gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa 3300

aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat 3360

ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa 3420

taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 3480

ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag 3540

agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 3600

ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 3660

taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 3720

cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 3780

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 3840

gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 3900

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 3960

ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 4020

cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 4080

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 4140

gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 4200

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 4260

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 4320

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 4380

aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 4440

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt 4500

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 4560

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 4620

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 4680

atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 4740

cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 4800

ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 4860

ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 4920

agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 4980

agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 5040

gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 5100

cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 5160

gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 5220

tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 5280

tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 5340

aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 5400

cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 5460

cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 5520

aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 5580

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 5640

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 5700

ccacctgacg tc 5712

<210> 5

<211> 5958

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 5

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta cccaccatgc caaagaagaa gcggaaggtc agtatgccgg 960

ataatcggca gccgaggaac cggcagccga ggatccgccc caatatggct gtgaaaagaa 1020

atcatagagg agagactttg ctccatattg cttctattaa gggcgacata ccttctgttg 1080

aatacctttt acaaaatgga agtgatccaa atgttaaaga ccatgctgga tggacaccat 1140

tgcatgaagc ttgcaatcat gggcacctga aggtagtgga attattgctc cagcataagg 1200

cattggtgaa caccaccggg tatcaaaatg actcaccact tcacgatgca gccaagaatg 1260

ggcatgtgga tatagtcaag ctgttacttt cctatggagc ctccagaaat gctgttaata 1320

tatttggtct gcggcctgtc gattatacag atgatgaaag tatgaaatcg ctattgctgc 1380

taccagagaa gaatgaatca tcctcagcta gccactgctc agtaatgaac actgggcagc 1440

gtagggatgg acctcttgta cttataggca gtgggctgtc ttcagaagat tacaaggatg 1500

acgacgataa gtgactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 1560

tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 1620

aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 1680

gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 1740

aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 1800

ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 1860

gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 1920

tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 1980

gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2040

attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 2100

cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 2160

ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 2220

aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 2280

agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 2340

ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 2400

catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 2460

aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 2520

agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg 2580

aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca 2640

agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 2700

ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 2760

tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 2820

cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac 2880

gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 2940

gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 3000

agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 3060

attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 3120

tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 3180

caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 3240

cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 3300

gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 3360

tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 3420

gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa 3480

atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc 3540

tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 3600

ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 3660

tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 3720

agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 3780

agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 3840

acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 3900

gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 3960

tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 4020

cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 4080

gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 4140

aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 4200

gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4260

aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 4320

gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 4380

ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 4440

cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 4500

ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 4560

actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4620

tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 4680

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4740

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 4800

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 4860

gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 4920

aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 4980

gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 5040

gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 5100

cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 5160

gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 5220

gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 5280

ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 5340

tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 5400

ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 5460

cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 5520

accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 5580

cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 5640

tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 5700

cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 5760

acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 5820

atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 5880

tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 5940

aaagtgccac ctgacgtc 5958

<210> 6

<211> 6132

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 6

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta cccaccatgc caaagaagaa gcggaaggtc agtatgccgg 960

ataatcggca gccgaggaac cggcagccga ggatccgcaa acctaggaaa agtttgttta 1020

atgatgcagg aaacaagaag aattcaatta aaatgtggtt tagccctcga agtaagaaag 1080

tcagatatgt tgtgagtaaa gcttcagtgc aaacccagcc tgcaataaaa aaagatatga 1140

agctgttgcc caatatggct gtgaaaagaa atcatagagg agagactttg ctccatattg 1200

cttctattaa gggcgacata ccttctgttg aatacctttt acaaaatgga agtgatccaa 1260

atgttaaaga ccatgctgga tggacaccat tgcatgaagc ttgcaatcat gggcacctga 1320

aggtagtgga attattgctc cagcataagg cattggtgaa caccaccggg tatcaaaatg 1380

actcaccact tcacgatgca gccaagaatg ggcatgtgga tatagtcaag ctgttacttt 1440

cctatggagc ctccagaaat gctgttaata tatttggtct gcggcctgtc gattatacag 1500

atgatgaaag tatgaaatcg ctattgctgc taccagagaa gaatgaatca tcctcagcta 1560

gccactgctc agtaatgaac actgggcagc gtagggatgg acctcttgta cttataggca 1620

gtgggctgtc ttcagaaaag cgacctgccg ccacaaagaa ggctggacag gctaagaaga 1680

agaaatgact cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt 1740

ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 1800

ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 1860

gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 1920

atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 1980

ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 2040

tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 2100

tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 2160

tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 2220

gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 2280

agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 2340

cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 2400

agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg 2460

tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 2520

agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 2580

tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc 2640

gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 2700

cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 2760

aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac 2820

aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc 2880

ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc 2940

cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc 3000

cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg 3060

cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt 3120

gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc 3180

catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga 3240

ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga 3300

tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct 3360

caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc 3420

gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt 3480

ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg 3540

cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat 3600

cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc 3660

gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa 3720

aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat 3780

ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa 3840

taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 3900

ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag 3960

agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 4020

ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 4080

taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 4140

cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 4200

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 4260

gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 4320

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 4380

ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 4440

cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 4500

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 4560

gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 4620

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 4680

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 4740

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 4800

aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 4860

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt 4920

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 4980

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 5040

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 5100

atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 5160

cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 5220

ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 5280

ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 5340

agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 5400

agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 5460

gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 5520

cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 5580

gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 5640

tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 5700

tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 5760

aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 5820

cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 5880

cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 5940

aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 6000

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 6060

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 6120

ccacctgacg tc 6132

<210> 7

<211> 7779

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 7

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta cccaccatgc caaagaagaa gcggaaggtc agtatgccgg 960

ataatcggca gccgaggaac cggcagccga ggatccgctc cgggaacgag cctcgttccg 1020

cgcccgccat ggaaccggat ggtcgcggtg cctgggccca cagtcgcgcc gcgctcgacc 1080

gcctggagaa gctgctgcgc tgctcgcgtt gtactaacat tctgagagag cctgtgtgtt 1140

taggaggatg tgagcacatc ttctgtagta attgtgtaag tgactgcatt ggaactggat 1200

gtccagtgtg ttacaccccg gcctggatac aagacttgaa gataaataga caactggaca 1260

gcatgattca actttgtagt aagcttcgaa atttgctaca tgacaatgag ctgtcagatt 1320

tgaaagaaga taaacctagg aaaagtttgt ttaatgatgc aggaaacaag aagaattcaa 1380

ttaaaatgtg gtttagccct cgaagtaaga aagtcagata tgttgtgagt aaagcttcag 1440

tgcaaaccca gcctgcaata aaaaaagatg caagtgctca gcaagactca tatgaatttg 1500

tttccccaag tcctcctgca gatgtttctg agagggctaa aaaggcttct gcaagatctg 1560

gaaaaaagca aaaaaagaaa actttagctg aaatcaacca aaaatggaat ttagaggcag 1620

aaaaagaaga tggtgaattt gactccaaag aggaatctaa gcaaaagctg gtatccttct 1680

gtagccaacc atctgttatc tccagtcctc agataaatgg tgaaatagac ttactagcaa 1740

gtggctcctt gacagaatct gaatgttttg gaagtttaac tgaagtctct ttaccattgg 1800

ctgagcaaat agagtctcca gacactaaga gcaggaatga agtagtgact cctgagaagg 1860

tctgcaaaaa ttatcttaca tctaagaaat ctttgccatt agaaaataat ggaaaacgtg 1920

gccatcacaa tagactttcc agtcccattt ctaagagatg tagaaccagc attctgagca 1980

ccagtggaga ttttgttaag caaacggtgc cctcagaaaa tataccattg cctgaatgtt 2040

cttcaccacc ttcatgcaaa cgtaaagttg gtggtacatc agggaggaaa aacagtaaca 2100

tgtccgatga attcattagt ctttcaccag gtacaccacc ttctacatta agtagttcaa 2160

gttacaggcg agtgatgtct agtccctcag caatgaagct gttgcccaat atggctgtga 2220

aaagaaatca tagaggagag actttgctcc atattgcttc tattaagggc gacatacctt 2280

ctgttgaata ccttttacaa aatggaagtg atccaaatgt taaagaccat gctggatgga 2340

caccattgca tgaagcttgc aatcatgggc acctgaaggt agtggaatta ttgctccagc 2400

ataaggcatt ggtgaacacc accgggtatc aaaatgactc accacttcac gatgcagcca 2460

agaatgggca tgtggatata gtcaagctgt tactttccta tggagcctcc agaaatgctg 2520

ttaatatatt tggtctgcgg cctgtcgatt atacagatga tgaaagtatg aaatcgctat 2580

tgctgctacc agagaagaat gaatcatcct cagctagcca ctgctcagta atgaacactg 2640

ggcagcgtag ggatggacct cttgtactta taggcagtgg gctgtcttca gaacaacaga 2700

aaatgctcag tgagcttgca gtaattctta aggctaaaaa atatactgag tttgacagta 2760

cagtaactca tgttgttgtt cctggtgatg cagttcaaag taccttgaag tgtatgcttg 2820

ggattctcaa tggatgctgg attctaaaat ttgaatgggt aaaagcatgt ctacgaagaa 2880

aagtatgtga acaggaagaa aagtatgaaa ttcctgaagg tccacgcaga agcaggctca 2940

acagagaaca gctgttgcca aagctgtttg atggatgcta cttctatttg tggggaacct 3000

tcaaacacca tccaaaggac aaccttatta agctcgtcac tgcaggtggg ggccagatcc 3060

tcagtagaaa gcccaagcca gacagtgacg tgactcagac catcaataca gtcgcatacc 3120

atgcgagacc cgattctgat cagcgcttct gcacacagta tatcatctat gaagatttgt 3180

gtaattatca cccagagagg gttcggcagg gcaaagtctg gaaggctcct tcgagctggt 3240

ttatagactg tgtgatgtcc tttgagttgc ttcctcttga cagcaagcga cctgccgcca 3300

caaagaaggc tggacaggct aagaagaaga aatgactcga gtctagaggg cccgtttaaa 3360

cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 3420

ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 3480

aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 3540

acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta 3600

tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta 3660

gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 3720

gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 3780

ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 3840

acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 3900

agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 3960

aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 4020

cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat 4080

tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag 4140

tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc 4200

agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct 4260

aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 4320

actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa 4380

gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat 4440

atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga 4500

tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 4560

acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 4620

ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc 4680

gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 4740

tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 4800

tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 4860

gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 4920

tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 4980

cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt 5040

cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 5100

attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 5160

ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 5220

tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 5280

agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat 5340

ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 5400

ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 5460

tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 5520

gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 5580

gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct 5640

gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 5700

aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 5760

gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 5820

agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 5880

gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 5940

gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 6000

cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 6060

aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 6120

tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 6180

ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 6240

ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 6300

cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 6360

ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 6420

gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 6480

atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 6540

aaacaaacca ccgctggtag cggttttttt gtttgcaagc agcagattac gcgcagaaaa 6600

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 6660

aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 6720

ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 6780

agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 6840

atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 6900

cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 6960

aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 7020

cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 7080

aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 7140

ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 7200

gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 7260

ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 7320

tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 7380

tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 7440

ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 7500

tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 7560

agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 7620

acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 7680

ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 7740

gttccgcgca catttccccg aaaagtgcca cctgacgtc 7779

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 8

aaacaagttc agcgtgtccg gcta 24

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 9

aaaatagccg gacacgctga actt 24

<210> 10

<211> 49

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 10

tataggcagt gggctgtctt cagaaatgga caagaagtac agcatcggc 49

<210> 11

<211> 56

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 11

taacgcgttc acttatcgtc atcgtctttg taatcgtcgc ctcccagctg agacag 56

<210> 12

<211> 29

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 12

gttctagaat gccaaagaag aagcggaag 29

<210> 13

<211> 49

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 13

gccgatgctg tacttcttgt ccatttctga agacagccca ctgcctata 49

<210> 14

<211> 10119

<212> DNA

<213> Artificial Sequence(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 14

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta cccaccatgc caaagaagaa gcggaaggtc agtatgccgg 960

ataatcggca gccgaggaac cggcagccga ggatccgcaa acctaggaaa agtttgttta 1020

atgatgcagg aaacaagaag aattcaatta aaatgtggtt tagccctcga agtaagaaag 1080

tcagatatgt tgtgagtaaa gcttcagtgc aaacccagcc tgcaataaaa aaagatccag 1140

agaagaatga atcatcctca gctagccact gctcagtaat gaacactggg cagcgtaggg 1200

atggacctct tgtacttata ggcagtgggc tgtcttcaga aatggacaag aagtacagca 1260

tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg 1320

tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc 1380

tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa 1440

ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca 1500

gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg 1560

tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg 1620

cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg 1680

acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc 1740

acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc 1800

agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg 1860

acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg 1920

cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg 1980

gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga 2040

gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg 2100

ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga 2160

gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg 2220

agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt 2280

acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag 2340

ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg 2400

aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca 2460

acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg 2520

aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc 2580

gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca 2640

gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt 2700

ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg 2760

tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag 2820

tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg 2880

ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg 2940

actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt 3000

tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc 3060

tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg 3120

aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag 3180

tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga 3240

tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg 3300

gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg 3360

acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc 3420

tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc 3480

tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga 3540

accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg 3600

gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc 3660

agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg 3720

aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc 3780

tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga 3840

gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc 3900

tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg 3960

gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga 4020

tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg 4080

acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc 4140

ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg 4200

cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg 4260

agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc 4320

aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca 4380

agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg 4440

gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc 4500

tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca 4560

aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg 4620

accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg 4680

ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca 4740

ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct 4800

acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg 4860

aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg 4920

ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg 4980

gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg 5040

acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc 5100

tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg 5160

agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact 5220

ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc 5280

tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag 5340

gcgacgatta caaggatgac gacgataagt gagaattcgc acatacatga ggatcaccca 5400

tgtctgcagc tcgagtctag aaaacatgag gatcacccat gtctgcagta ttcccgggtt 5460

cattagatcc taaggtacct aattgcctag aaaacatgag gatcacccat gtctgcagct 5520

cgagtctaga aaacatgagg atcacccatg tctgcagtat tcccgggttc attagatcct 5580

aaggtaccta attgcctaga aaacatgagg atcacccatg tctgcagctc gagtctagaa 5640

aacatgagga tcacccatgt gtgcaccttg tcgacctcga gtctagaggg cccgtttaaa 5700

cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 5760

ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 5820

aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 5880

acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta 5940

tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta 6000

gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 6060

gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 6120

ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 6180

acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 6240

agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 6300

aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 6360

cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat 6420

tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag 6480

tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc 6540

agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct 6600

aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 6660

actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa 6720

gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat 6780

atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga 6840

tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 6900

acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 6960

ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc 7020

gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 7080

tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 7140

tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 7200

gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 7260

tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 7320

cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt 7380

cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 7440

attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 7500

ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 7560

tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 7620

agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat 7680

ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 7740

ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 7800

tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 7860

gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 7920

gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct 7980

gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 8040

aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 8100

gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 8160

agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 8220

gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 8280

gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 8340

cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 8400

aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 8460

tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 8520

ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 8580

ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 8640

cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 8700

ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 8760

gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 8820

atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 8880

aaacaaacca ccgctggtag cggttttttt gtttgcaagc agcagattac gcgcagaaaa 8940

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 9000

aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 9060

ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 9120

agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 9180

atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 9240

cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 9300

aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 9360

cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 9420

aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 9480

ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 9540

gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 9600

ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 9660

tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 9720

tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 9780

ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 9840

tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 9900

agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 9960

acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 10020

ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 10080

gttccgcgca catttccccg aaaagtgcca cctgacgtc 10119

Claims (10)

1. An accessory protein for improving DNA repair efficiency is characterized in that the nucleotide sequence of the accessory protein is shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

2. An expression plasmid comprising the helper protein of claim 1.

3. The expression plasmid of claim 2, wherein the nucleotide sequence of the expression plasmid is set forth as SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6.

4. A gene editing vector for increasing the efficiency of DNA repair comprising the helper protein of claim 1, a DNA editing protein, and a delivery vector.

5. The gene editing vector of claim 4, wherein the DNA editing protein comprises a Cas9 protein, a CasX protein, a Cas Φ protein, a Cas12 protein, a Cas14 protein.

6. The gene editing vector of claim 5, wherein the accessory protein is fused to the carbon terminus or nitrogen terminus of the DNA editing protein.

7. The gene editing vector of claim 6, wherein the delivery vector comprises a viroid vector, a Lentivirus, an adenovirus vector AAV, a retropmscv virus, a pCMV cloning vector, and an integration-defective viroid vector.

8. The gene editing vector of claim 7, wherein the integration defective viroid vector system comprises psPAX2-D64V-NC-MS2, pCMV-BRID-Cas9-6XMS2 plasmid.

9. Use of the accessory protein of claim 1, the expression plasmid of claim 2 or 3, or the gene editing vector of any one of claims 4 to 8 for the preparation of a product for increasing DNA repair efficiency.

10. Use according to claim 9, wherein the DNA repair is by homologous recombination.

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