CN108715864A - It is a kind of for the gene insertion mutation system and its mutation method of mouse embryo stem cell and application - Google Patents

Abstract

The invention discloses a kind of for the gene insertion mutation system and its mutation method of mouse embryo stem cell and application.The present invention constructs the endogenous gene mutational vector containing riddled basins and inducible mutational vector carries out the transposase expression vector of genome conformity, utilize electrotransfection method, two kinds of carriers are imported in mouse embryo stem cell jointly, it is analyzed again by inducing transposition expression of enzymes, cell resistance screening, cell clone identification and integration site, effective mutation method of mouse embryo stem cell endogenous gene is built, the cell clone for carrying single mutator and having totipotential differentiation ability is obtained.Mutational vector of the present invention can interfere the transcriptional expression of the endogenous gene in the genome of radom insertion mouse embryo stem cell.Mutation method of the present invention can carry out extensive insertion mutation to mouse embryo stem cell genome, obtain the cell clone library for carrying single mutator and having totipotential differentiation ability, the development for mouse mutant and gene functional research.

Description

A gene insertion mutation system and mutation method for mouse embryonic stem cells and application

technical field

The invention relates to the gene insertion mutation cloning and application technology of mouse embryonic stem cells, more specifically, the invention relates to a gene insertion mutation system for mouse embryonic stem cells and its mutation method and application.

Background technique

An important means of gene function research is to use genetic manipulation techniques to overexpress or knock out/down genes in model animals such as mice, zebrafish, and fruit flies, and to observe abnormal phenotypes in life processes such as embryonic development and organ formation. , to analyze the molecular mechanism of genes in embryonic development, organ formation, and disease occurrence. As a mammalian model organism, mice have incomparable advantages in the study of human gene functions, but mouse live gene targeting technology is time-consuming and laborious, which seriously affects the process of gene function research. Mouse embryonic stem cells are favored by researchers for their ability to self-renew and differentiate into the three germ layers. Over the past two decades, the International Association for Mouse Research has been committed to mouse embryonic stem cell gene trapping and gene targeting mutation screening. As of January 2018, the International Gene Trapping Consortium has obtained 121,703 mouse embryonic stem cell clones using gene trapping methods. 12393 genes were captured. In order to realize the mutation research at the whole gene level of mice, different gene trapping and gene targeting technologies need to be used. The gene trapping vectors reported so far have played an important role in mouse gene trapping and gene function research, but there are also some shortcomings , such as the preference of gene insertion sites, the effectiveness of gene capture, etc. According to the needs of gene function research, combined with the transposition advantage of SB transposon in mammals, we constructed a polyA gene trap vector based on Sleeping Beauty (SB) transposon, in order to explore more functional genes.

Contents of the invention

The purpose of the present invention is to provide a mouse embryonic stem cell gene insertion mutation system and its mutation method and application. The gene insertion mutation system of the present invention can be randomly inserted into the genome of the mouse embryonic stem cell, and the insertion mutation of the mouse embryonic stem cell gene is helpful for researchers to conduct functional research on the mouse gene.

The first aspect of the present invention provides a sleeping beauty SB transposon-mediated polyA gene trapping vector, which includes: SA-EXON-SV40 polyA-TT gene mutation element, SV40 promoter, IRES - SD-ARE sequence, screening marker gene Neo, FRT sequence, gene in series in the order of SA-EXON-SV40PolyA-TT-FRT-SV40-Neo-FRT-IRES-SD-ARE.

The construction method of the gene trap vector described in the above technical scheme is as follows: the pT2/polyA-trap vector is subjected to AatII and SacII double enzyme digestion, and the SA-EXON-SV40polyA-TT gene mutation element, SV40 promoter, screening marker gene Neo , IRES-SD-ARE sequence, were connected to obtain the pT2/SA-EXON-SV40PolyA-TT-SV40-Neo-IRES-SD-ARE vector, and then the FRT sequence was cloned to both ends of SV40-Neo by PCR method to obtain the The gene trap vector pT2/SA-EXON-SV40PolyA-TT-FRT-SV40-Neo-FRT-IRES-SD-ARE, the nucleic acid sequence of the gene trap vector is shown in SEQ ID No.1.

The second aspect of the present invention provides an inducible transposase expression vector, the transposase expression vector includes CMV promoter, Tet-on system (rtTA and TRE elements), SB11 transposase, βactin polyA sequence, the gene is connected in series in the order of pCMV-rtTA-TRE-SB11-βactin polyA.

The construction method of the transposase expression vector described in the above technical scheme is as follows:

The TRE-miniCMV fragment was cloned into the vector PCMV-rtTA-SB11-βactin polyA by digesting the AgeI/BsiWI double restriction site to obtain the transposase expression vector PCMV-rtTA-TRE-SB11-βactin polyA. The nucleic acid sequence of the vector is shown in SEQ ID No.2.

The above-mentioned transposase expression vector of the present invention induces the expression of SB11 transposase only in the presence of DOX, and the SB11 transposase binds to the SB transposon carrier, and inserts the capture vector into the genome through a transposition mechanism.

A third aspect of the present invention provides a gene insertion mutation system for mouse embryonic stem cells, the gene insertion mutation system comprising:

(1) the polyA gene trapping vector mediated based on Sleeping Beauty SB transposon; and

(2) The inducible transposase expression vector.

A fourth aspect of the present invention provides the above-mentioned gene insertion mutation method of mouse embryonic stem cells, said method comprising the steps of:

(a) Construction of an insertion mutagenesis system, including the construction of the polyA gene trap vector mediated by the Sleeping Beauty SB transposon and the construction of the transposase expression vector that controls the function of the gene trap vector;

(b) Genetic transformation of mouse embryonic stem cells and screening of cell clones;

(c) Analysis of integration sites of embryonic stem cell insertion mutation clones;

(d) Expression analysis of mutant genes.

Further, in step (b) of the above technical solution, the genetic transformation of introducing the mutant vector into embryonic stem cells is specifically electroporation, and the specific electroporation process is as follows: digest and collect mouse ES cells, the number of which is ¹ ×107 cells, and the amount of mutant vector DNA used is 25 μg , using a Bio-rad electroporation instrument for electroporation, the parameters are: voltage 250, capacitance 500, resistance ∞, electroporation cup diameter 4mm, wherein: the mutation vector includes a gene capture vector and a transposase expression vector, and the mass ratio of the two is 1: 1.

Further, the insertion mutation clone screening and insertion site analysis in the above technical scheme, the specific process is as follows: After the cells are electroporated, 1 μg/ml Dox is added to the culture medium to induce the expression of SB11 transposase, and after 24 hours, 200 μg/ml Dox is used to induce the expression of SB11 transposase. Select transfected cells with ml G418, replace the medium once a day, and continue the screening for 1 week. After expanding the surviving mES monoclonal clones, select different cell clones for positive clone screening and insertion site analysis, and sequence the obtained fragments. Database Alignment analysis obtained capture gene loci.

Preferably, the positive screening primer sequence described in the above technical scheme includes:

Primer F: 5'-GACCACCAAGCGAAACATCG-3' (SEQ ID No.3);

Primer R: 5'-ATATCACGGGTAGCCAACGC-3' (SEQ ID No. 4).

Preferably, the insertion site analysis described in the above technical solution specifically uses the Splinker PCR method for insertion site analysis.

Further, the expression analysis of the mutated gene described in step (d) of the technical solution includes analysis of fusion transcripts, fluorescent quantitative PCR and Southern blot analysis.

The fifth aspect of the present invention provides the application of the above-mentioned gene insertion mutation method, which is mainly used for performing large-scale gene insertion mutation on the mouse embryonic stem cell genome, screening single-cell clone libraries of different gene insertion mutations in batches, and obtaining Single mutant gene and cell clone library with totipotent differentiation ability are used for the development of mouse mutants and gene function research.

Furthermore, the present invention analyzes the totipotent differentiation ability of cell clones carrying a single gene mutation and establishes a single-cell clone library, including the expression detection of embryonic stem cell marker genes of cell clones and batch screening of single-cell clones. Immunofluorescence staining was performed using antibodies against molecular marker genes SSEA-1 and OCT-4. At the same time, fluorescent quantitative PCR was used to detect the expression levels of molecular marker genes SSEA-1, OCT-4, REX-1 and NANOG, and alkaline phosphoric acid Enzyme staining and chromosomal karyotype analysis confirmed that the cell clones obtained by the mutation method had the totipotency of embryonic stem cells. Molecular marker detection and karyotype analysis showed that the mutant mouse ES cell clones we obtained had high gonadal chimerism, which laid the foundation for further use of them to prepare genetically modified mice.

The specific process of developing mouse mutants using cell clones carrying a single mutant gene described in the above-mentioned technical scheme is as follows. By blastocyst microinjection, the embryonic stem cell line with normal karyotype is injected into the C57BL/6J mouse In blastocysts, chimeric mice were obtained, and then the mice with high chimerism were selected to be crossed with C57BL/6J mice to obtain heterozygous mice carrying the mutant gene, and then self-crossed to obtain homozygous mutant mice.

Compared with the prior art, the gene insertion mutation system for mouse embryonic stem cells and its mutation method and application of the present invention have the following beneficial effects:

(1) The gene insertion mutation system of the present invention includes polyA gene trapping vectors mediated by SB transposons and inducible transposase expression vectors, and the present invention utilizes the Sleeping Beauty transposon system to mediate gene mutation elements and gene trapping The element is used for genome integration mutation of mouse embryonic stem cells, which can realize rapid and effective mutation of mES genes, and help to explore the function of genes in embryonic development and disease process.

(2) The gene mutation method of mouse embryonic stem cells of the present invention includes the construction of an insertion mutation system based on Sleeping Beauty transposon, genetic transformation and screening of mouse embryonic stem cells, integration site analysis of embryonic stem cell insertion mutation clones, and gene expression analysis Wait. The purpose of the gene mutation method is to overcome the deficiency of the gene trapping carrier in the existing mouse embryonic stem cells and provide an effective gene mutation method.

Description of drawings

Figure 1 is a schematic diagram of the construction of the polyA gene capture vector mediated by the Sleeping Beauty SB transposon described in Example 1 of the present invention;

Figure 2 is a schematic diagram of the construction of the inducible transposase expression vector described in Example 2 of the present invention;

3 is a schematic diagram of the electroporation and insertion mutation clone screening process described in Example 3 and Example 4 of the present invention;

Fig. 4 is the expression analysis diagram of the captured gene described in Example 5 of the present invention; wherein: A-gene capture vector insertion genome site schematic diagram, B-gene capture vector and endogenous gene fusion expression analysis primer position schematic diagram, C-gene Capture vector and endogenous gene fusion expression analysis electropherogram, D-capture gene expression analysis, E-Southern blot analysis gene capture vector genome copy number;

Fig. 5 is the cell pluripotency analysis diagram described in Example 6 of the present invention, wherein: A-immunofluorescence staining detects ES marker gene expression, B-fluorescence quantitative PCR detects ES marker gene expression, C-ES cell clone alkaline phosphate Enzyme activity analysis, D-ES cell clone chromosome karyotype analysis.

Detailed ways

The technical solution of the present invention will be described in further detail below through specific embodiments and accompanying drawings. The following embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art may use the technical content disclosed above to change into equivalent embodiments with equal changes. Any simple modifications or equivalent changes made to the following embodiments according to the technical essence of the present invention without departing from the solution content of the present invention fall within the protection scope of the present invention.

The experimental methods in the various embodiments of the present invention are all carried out under conventional conditions, specifically referring to the experimental methods described in Sambrook et al., Molecular Cloning Experiment Guide (Second Edition, 1992, Science Press).

In the present invention, by constructing a gene mutation carrier containing screening marker gene expression and an inducible mediating gene mutation carrier for genome integration, the two vectors are co-introduced into mouse embryonic stem cells by electrotransfection method, and the expression vector is induced to express , Cell resistance screening and cell clone identification analysis, construct an effective mutation method for mouse embryonic stem cell genes, and obtain cell clones carrying a single mutant gene and possessing totipotent differentiation ability.

The mutation carrier of the present invention can be randomly inserted into the genome of mouse embryonic stem cells, including exons, introns and non-coding regions. Using this method, the mutant vector is inserted into the intron of the mouse gene X to interfere with the transcription and expression of the endogenous gene. This method can be randomly inserted into the genome of mouse embryonic stem cells. Using this method, large-scale gene insertion mutations can be performed on the genome of mouse embryonic stem cells, and a cell clone library carrying a single mutant gene and having the ability to differentiate can be obtained and used for mouse mutations. Body development and gene function studies.

Example 1

A polyA gene trap vector mediated by Sleeping Beauty SB transposon in this embodiment, said gene trap vector includes: SA-EXON-SV40 polyA-TT gene mutation element, SV40 promoter, IRES-SD-ARE sequence, Screening marker gene Neo, FRT sequence, gene in series in the order of SA-EXON-SV40PolyA-TT-FRT-SV40-Neo-FRT-IRES-SD-ARE.

The method for constructing the polyA gene trapping vector mediated by the sleeping beauty SB transposon described above is as follows:

As shown in Figure 1, the pT2/polyA-trap vector (Song, Li et al.2012) was digested with AatII and SacII, and the SA-EXON-SV40polyA-TT gene mutation element, SV40 promoter, IRES-SD- ARE sequence, the screening marker gene Neo was connected to obtain the pT2/SA-EXON-SV40PolyA-TT-SV40-Neo-IRES-SD-ARE vector, and then the FRT sequence was cloned to both ends of SV40-Neo by PCR method to obtain the capture vector pT2/SA-EXON-SV40PolyA-TT-FRT-SV40-Neo-FRT-IRES-SD-ARE, the nucleic acid sequence of the capture vector is shown in SEQ ID No.1.

Example 2

A kind of inducible transposase expression vector of the present embodiment, described transposase expression vector comprises CMV promoter, Tet-on system (rtTA and TRE element), SB11 transposase, βactin polyA sequence, gene presses PCMV Sequential concatenation of -rtTA-TRE-SB11-βactin polyA.

The construction method of the above-mentioned transposase expression vector is as follows:

As shown in Figure 2, the TRE-miniCMV fragment was cloned into the vector pCMV-rtTA-SB11-βactin polyA by digesting the AgeI/BsiWI double restriction site to obtain the transposase expression vector pCMV-rtTA-TRE- SB11-βactin polyA, the nucleic acid sequence of the transposase expression vector is shown in SEQ ID No.2.

The transposase expression vector described above in this example can only induce the expression of SB11 transposase in the presence of DOX, and the SB11 transposase is bound to the SB transposon vector, and the capture vector is inserted into the genome through the transposition mechanism .

Example 3 Mouse Embryonic Stem Cell Culture and Electrotransformation

Mouse embryonic stem cell (mES) line We use R1/E cell line (ATCC: SCRC-1036 ^TM ), the culture conditions are: 37°C, 5% carbon dioxide, ES complete medium components: DMEM (invitrogen 12430), ES Grade FBS (biochromS4115), Glutamax (invitrogen 35050), NEAA (invitrogen 11140), LIF (MilliporeESG1107), β-Mer (invitrogen 21985).

The feeder cells used for R1/E in this example: MEF-G418 (Kunming Bai).

In this example, the mutant vector (including gene trapping vector and transposase expression vector, the mass ratio of the two is 1:1) is introduced into the electrotransformation of mouse embryonic stem cells, the specific process is shown in Figure 3: mouse ES cells are digested and collected, The quantity is 1×10 ⁷ cells, the amount of mutation vector DNA (including gene trap vector and transposase expression vector, the mass ratio of the two is 1:1) is 25 μg, and the electroporation is performed using a Bio-rad electroporator (parameters: voltage 250, Capacitance 500, resistance ∞, electric cup diameter 4mm).

Example 4 Gene trap site cloning

As shown in Figure 3, after the cells were electroporated, 1 μg/ml Dox was added to the culture medium to induce the expression of SB11 transposase. After 24 hours, the transfected cells were screened with 200 μg/ml G418, and the medium was changed once a day, and the screening lasted for 1 hour. Zhou, after expanding the culture of the surviving mES monoclonal, select different cell clones for positive clone screening and insertion site analysis, positive screening primer sequence:

Primer F: 5'-GACCACCAAGCGAAACATCG-3' (SEQ ID No.3);

Primer R: 5'-ATATCACGGGTAGCCAACGC-3' (SEQ ID No. 4).

The insertion site was analyzed by Splinker PCR method, the obtained fragment was sequenced, and the captured gene site was obtained by database comparison analysis. The primers used are as follows:

Long stand: 5'-CGAAGAGTAACCGTTGCTAGGAGAGACCGTGGCTGAATGAGACTGGT

GTCGACACTAGTGG-3' (SEQ ID No. 5);

Short Sau3aI: 5'-GATCCCACTAGTGTCGACACCAGTCTCTAATTTTTTTTTTCAAAAAAAA-3' (SEQ ID No. 6);

Splink1: 5'-CGAAGAGTAACCGTTGCTAGGAGAGACC-3' (SEQ ID No.7);

Splink2: 5'-GTGGCTGAATGAGACTGGTGTCGAC-3' (SEQ ID No.8);

L2: 5'-TTAAAGGCACAGTCAACTTAGTGTATGTAAACTTCTG-3' (SEQ ID No. 9);

L3: 5'-TGAAAAACGAGTTTTAATGACTCCAACTTAAG-3' (SEQ ID No. 10);

R1: 5'-CTAACTGACCTAAGACAGGGAATTTTTACTAGGATT-3' (SEQ ID No. 11);

R2: 5'-TAAGGTGTATGTAAACTTCCGACTTCAACTG-3' (SEQ ID No. 12).

Table 1 is the gene trap site analysis diagram of Example 4 of the present invention

Example 5 capture gene expression analysis

Gene expression analysis includes fusion transcript analysis, fluorescent quantitative PCR and Southern blot analysis. One of the cell clones obtained by our screening was inserted into the third intron of the syngap1 gene (Figure 4A), and we named the clone as syngap1 ^+/- cell clone, using primer X (Figure 4B), including:

F: 5'-ACACCCCCTCTGATCCGCG-3' (SEQ ID No.13);

R', 5'-CGGCCGCTCGTACGTCACTAATTC-3' (SEQ ID No.14) and primer sequences were used for transcript fusion expression analysis, and primer X confirmed the fusion transcription of SA-EXON and exon 3 of syngap1 gene in the capture vector (Fig. 4C). Then we quantified the expression of the endogenous gene syngap1, and the quantitative primer sequences are as follows:

F: 5'-ACACCCCCTCTGATCCGCG-3' (SEQ ID No.13);

R: 5'-TCCATGCTGTACTGTTTCCCCCCC-3' (SEQ ID No. 15) It was found that in syngap1 ^+/- cell clones, the expression of syngap1 gene decreased by about 50% ( FIG. 4D ). The genome of the syngap1 ^+/- cell clone was digested with BglII and EcoRI, and analyzed by Southern blot. It was found that there was one copy of the capture vector in the genome, which was consistent with the expression level of the syngap1 gene (Fig. 4E). The above results indicate that in ES cells, the capture vector can effectively knock down and mutate endogenous genes.

Example 6 Cell pluripotency analysis.

Immunofluorescence staining was used to detect the expression of ES marker genes SSEA-1 and OCT-4, and the results of fluorescent color development showed that SSEA-1 and OCT-4 were expressed in the syngap1 ^+/- cell clone, which confirmed that the syngap1 ^+/- cell clone had small Totipotent activity of mouse embryonic stem cells. The specific operation process is as follows,

(1) Cells were grown in 24-well plates and washed with PBS;

(2) Fix with 4% paraformaldehyde for 20 minutes, wash with PBS;

(3) 0.5% Triton X-100 permeate the membrane for 10 minutes, wash with 0.1% BSA;

(4) Block at 37°C for 30 minutes, (blocking solution 10% goat serum + 0.1% Triton X-100 + 1% BSA);

(5) Incubate primary antibody SSEA-1 20 μg/ml (R&D Systems, USA) and OCT-4 20 μg/ml (Abcam, UK), overnight at 4°C;

(6) Wash with 0.1% BSA;

(7) Incubate the secondary antibody at 37°C for 30 minutes, goat anti-mouse-Cy3 and goat anti-rabbit-FITC (BOSTER, China);

(8) Wash with 0.1% BSA;

(9) Counterstain DAPI for 5 minutes at room temperature;

(10) Fluorescence microscope observation and photography (Leica DMI6000B/DMi8).

The mouse embryonic stem cell marker genes were quantitatively detected by fluorescent quantitative PCR. The quantitative PCR results showed that the expression of stem cell marker genes SSEA-1, OCT-4, Rex-1, and NANOG in syngap1 ^+/- cell clones were not affected. Syngap1 ^+/- cell clones were confirmed to have mouse embryonic stem cell totipotent activity (Fig. 5B). The required primer sequences are as follows:

ssea1f3: 5'-CGGACCGACTCGGATGTC-3' (SEQ ID No. 16);

ssea1r3: 5'-CCGACTCAGCTGGTGGTAGTAA-3' (SEQ ID No. 17);

oct4f: 5'-TTCCCTCTGTTCCCGTCACT-3' (SEQ ID No. 18);

oct4r: 5'-TGGTGCCTCAGTTTGAATGC-3' (SEQ ID No. 19);

Rex-1f: 5'-CCGTATCAGTGCACGTTCGA-3' (SEQ ID No. 20);

Rex-1r: TGGGTGCGCAAGTTGAAA-3' (SEQ ID No. 21);

Nanog f: 5'-CCTCATCAATGCCTGCAGTTT-3' (SEQ ID No.22);

Nanog r: 5'-CTCAGTAGCAGACCCTTGTAAGCA-3' (SEQ ID No. 23);

GAPDH f: 5'-ACATCATCCCTGCATCCACT-3' (SEQ ID No. 24);

GAPDH r: 5'-AGATCCACGACGGACACATT-3' (SEQ ID No. 25);

After the mouse embryonic stem cells were cultured for 5 days, they were tested according to the instructions of the alkaline phosphatase detection kit. The test results showed that syngap1 ^+/- had alkaline phosphatase activity, while no alkaline phosphatase activity was detected in the negative control MEF cells ( Figure 5C).

Karyotype analysis showed that syngap1 ^+/- cell clones had normal XY-type chromosomes (Fig. 5D), indicating that the mutant mouse ES cell clones we obtained had high gonadal chimerism, and we could use them to prepare genetically modified small Rats laid the groundwork. Chromosomal and sex analysis procedures are as follows:

Take a T25 flask full of cells that is more than 90% confluent, and add colchicine 3 hours in advance;

(1) KCl hypotonic solution in warm bath at 37°C, pre-cooled methanol-glacial acetic acid fixative solution;

(2) Digest the cells, pipette evenly, transfer to a 60mm culture dish, and gently tilt the culture dish after adhering to the wall at 37°C for 1 hour, take the supernatant, and transfer it into a 15ml centrifuge tube;

(3) Centrifuge at 12000rpm for 5 minutes;

(4) Remove the supernatant, leave a small amount of residual liquid and mix well, add 8ml of warmed KCl hypotonic solution, mix upside down, warm bath at 37°C for 30 minutes, mix up and down once in the middle;

(5) Add 1ml of pre-cooled methanol-glacial acetic acid fixative solution to stop hypotonicity, mix upside down, and centrifuge at 1400rpm for 3 minutes;

(6) Remove the supernatant, leave a small amount of residual liquid to resuspend the cells, add 10ml of pre-cooled methanol-glacial acetic acid fixative, mix up and down, place at room temperature for 20 minutes, and mix up and down once in the middle;

(7) Centrifuge at 1200rpm for 5 minutes, repeat step (7) twice, and place at room temperature for 10 minutes for the second time;

(8) During the waiting period, take an appropriate amount of slides soaked in absolute ethanol, wash with double distilled water, put the slides into a beaker filled with boiling water, and heat the slides;

(9) Centrifuge at 1200rpm for 5 minutes, remove the supernatant, and leave a small amount of residual liquid to resuspend the cells;

(10) Put the glass slide on the ground, put an appropriate amount of absorbent paper under it, take an appropriate amount of cell suspension, drop it at a certain height, then take the glass slide and bake it on an alcohol lamp, and dry the slide overnight;

(11) Giemsa staining for 1 hour, wash the slides, observe and take pictures under the microscope.

sequence listing

<110> Institute of Hydrobiology, Chinese Academy of Sciences

<120> A gene insertion mutation system for mouse embryonic stem cells and its mutation method and application

<130> None

<160> 25

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5968

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 60

ccagtgagcg cgcgtaatac gactcactat agggcgaatt ggagctcgga tccctataca 120

gttgaagtcg gaagtttaca tacacttaag ttggagtcat taaaactcgt ttttcaacta 180

ctccacaaat ttcttgttaa caaacaatag ttttggcaag tcagttagga catctacttt 240

gtgcatgaca caagtcattt ttccaacaat tgtttacaga cagattattt cacttataat 300

tcactgtatc acaattccag tgggtcagaa gtttacatac actaagttga ctgtgccttt 360

aaacagcttg gaaaattcca gaaaatgatg tcatggcttt agaagcttgc tagcgattgc 420

agcacgaaac aggaagctga ctccacatgg tcacatgctc actgaagtgt tgacttccct 480

gacagctgtg cactttctaa accggttttc tcattcattt acagttcagc cgatgatgaa 540

attgccgcac tggttgttag caacgtagcc ggtatgtgaa tgatgaatta gtgacgtacg 600

agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt 660

taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg 720

ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 780

caaataaagc atttttttca ctgcgaattc tttttatttt atttttttt taattttttt 840

ttgacgtcga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttcccag 900

gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc 960

ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 1020

gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 1080

ccccatggct gactaatttt ttttttat gcagaggccg aggccgcctc ggcctctgag 1140

ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg 1200

ggagcttgga tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg 1260

attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 1320

tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 1380

caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 1440

gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 1500

gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 1560

ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 1620

cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 1680

gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 1740

catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc 1800

gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 1860

cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 1920

gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 1980

gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 2040

gagttcttct gagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc 2100

actagtgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag 2160

gccggtgtgc gtttgtctat atgttatttt ccaccacatatt gccgtctttt ggcaatgtga 2220

gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg 2280

ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt 2340

gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca 2400

ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaaccccc 2460

agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat 2520

tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc 2580

ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga 2640

accacgggga cgtggttttc ctttgaaaaa cacgatgata atatcgccac aaccatggac 2700

catgcaccat gtacaaaccc ccccaaaccg aaggtgagtt gatctttaag ctttttacat 2760

tttcagctcg catatatcaa ttcgaacgtt taattagaat gtttaaataa agctagatta 2820

aatgattagg ctcagttacc ggtctttttt ttctcattta cgtagatcta gcttgtggaa 2880

ggctactcga aatgtttgac ccaagttaaa caatttaaag gcaatgctac caaatactaa 2940

ttgagtgtat gtaaacttct gacccactgg gaatgtgatg aaagaaataa aagctgaaat 3000

gaatcattct ctctactatt attctgatat ttcacattct taaaataaag tggtgatcct 3060

aactgaccta aagacagggaa tttttactag gattaaatgt caggaattgt gaaaaagtga 3120

gtttaaatgt atttggctaa ggtgtatgta aacttccgac ttcaactgta tagggatcct 3180

ctagctagag tcgacctcga gggggggccc ggtacccagc ttttgttccc tttagtgagg 3240

gttaatttcg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 3300

gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 3360

atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3420

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3480

tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 3540

agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 3600

aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 3660

gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 3720

tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 3780

cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 3840

ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 3900

cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 3960

atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 4020

agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 4080

gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 4140

gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 4200

tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 4260

agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 4320

gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 4380

aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 4440

aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 4500

ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 4560

gataccgcga gacccacgct caccggctcc agattatca gcaataaacc agccagccgg 4620

aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 4680

ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 4740

tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 4800

ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 4860

cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 4920

agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 4980

gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 5040

gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 5100

acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 5160

accactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 5220

agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 5280

aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 5340

gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 5400

tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 5460

ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 5520

cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 5580

ccctttagg ttccgatta gtgctttacg gcacctcgac cccaaaaaac ttgattagggg 5640

tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 5700

gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 5760

ggtctattct tttgattatt aagggatttt gccgatttcg gcctattggt taaaaaatga 5820

gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttccat 5880

tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 5940

cgccagctgg cgaaaggggg atgtgctg 5968

<210> 2

<211> 6673

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 60

ccagtgagcg cgcgtaatac gactcactat agggcgaatt ggagctcgga tccgctagca 120

tatatgacgt cataacttcg tatagcatac attatacgaa gttatgttga cattgattat 180

tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 240

tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 300

cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 360

gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 420

tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 480

agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 540

ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac 600

ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 660

aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc 720

gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga 780

gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgcg 840

gccccgaatt caccatgtct agactggaca agagcaaagt cataaacggc gctctggaat 900

tactcaatgg agtcggtatc gaaggcctga cgacaaggaa actcgctcaa aagctgggag 960

ttgagcagcc taccctgtac tggcacgtga agaacaagcg ggccctgctc gatgccctgc 1020

caatcgagat gctggacagg catcataccc acttctgccc cctggaaggc gagtcatggc 1080

aagactttct gcggaacaac gccaagtcat tccgctgtgc tctcctctca catcgcgacg 1140

gggctaaagt gcatctcggc acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc 1200

agctcgcgtt cctgtgtcag caaggcttct ccctggagaa cgcactgtac gctctgtccg 1260

ccgtgggcca ctttacactg ggctgcgtat tggaggaaca ggagcatcaa gtagcaaaag 1320

aggaaagaga gacacctacc accgattcta tgcccccact tctgagacaa gcaattgagc 1380

tgttcgaccg gcagggagcc gaacctgcct tccttttcgg cctggaacta atcatatgtg 1440

gcctggagaa acagctaaag tgcgaaagcg gcgggccggc cgacgccctt gacgattttg 1500

acttagacat gctcccagcc gatgcccttg acgactttga ccttgatatg ctgcctgctg 1560

acgctcttga cgattttgac cttgacatgc tccccgggta actaagtaag gatccagaca 1620

tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct 1680

ttatttgtga aatttgtgat gctattgctt tatttgtaac catttataagc tgcaataaac 1740

aagttgctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 1800

gccctttcgt cttcactcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc 1860

gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat 1920

cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg 1980

aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact 2040

ccctatcagt gtagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga 2100

aaagtgaaag tcgagctcgg tacccgggtc gagtaggcgt gtacggtggg aggcctatat 2160

aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga 2220

cctccataga agacaccggg accgatccag cctcctagga ccggtgccac catgggaaaa 2280

tcaaaagaaa tcagccaaga cctcagaaaa aaaattgtag acctccacaa gtctggttca 2340

tccttgggag caatttccaa acgcctgaaa gtaccacgtt catctgtaca aacaatagta 2400

cgcaagtata aacaccatgg gaccacgcag ccgtcatacc gctcaggaag gagacgcgtt 2460

ctgtctccta gagatgaacg tactttggtg cgaaaagtgc aaatcaatcc cagaacaaca 2520

gcaaaggacc ttgtgaagat gctggaggaa acaggtacaa aagtatctat atccacagta 2580

aaacgagtcc tatatcgaca taacctgaaa ggccgctcag caaggaagaa gccactgctc 2640

caaaaccgac ataagaaagc cagactacgg tttgcaagag cacatgggga caaagatcgt 2700

actttttgga gaaatgtcct ctggtctgat gaaacaaaaa tagaactgtt tggccataat 2760

gaccatcgtt atgtttggag gaagaagggg gaggcttgca agccgaagaa caccatccca 2820

accgtgaagc acgggggtgg cagcatcatg ttgtgggggt gctttgctgc aggagggact 2880

ggtgcacttc acaaaataga tggcatcatg aggaaggaaa attatgtgga tatattgaag 2940

caacatctca agacatcagt caggaagtta aagcttggtc gcaaatgggt cttccaacaa 3000

gacaatgacc ccaagcatac ttccaaacac gtgagaaaat ggcttaagga caacaaagtc 3060

aaggtattgg agtggccatc acaaagccct gacctcaatc ctatagaaaa tttgtgggca 3120

gaactgaaaa agcgtgtgcg agcaaggagg cctacaaacc tgactcagtt acaccagctc 3180

tgtcaggagg aatgggccaa aattcaccca acttattgtg ggaagcttgt ggaaggctac 3240

ccgaaacgtt tgacccaagt taaacaattt aaaggcaatg ctaccaaata ctaggcggcc 3300

gcctatcgat aaacggactg ttaccacttc acgccgactc aactgcgcag agaaaaactt 3360

caaacgacaa cattggcatg gcttttgtta tttttggcgc ttgactcagg atctaaaaac 3420

tggaacggcg aaggtgacgg caatgttttg gcaaataagc atccccgaag ttctacaatg 3480

catctgagga ctcaatgtttttttttttttttttttcttt agtcattcca aatgtttgtt 3540

aaatgcattg ttccgaaact tattgcctc tatgaaggct gcccagtaat tgggagcata 3600

cttaacattg tagtattgta tgtaaattat gtaacaaaac aatgactggg tttttgtact 3660

ttcagcctta atcttgggttttttttttttttttttggtt ccaaaaaact aagagctctt 3720

taccattcaa gatgtaaagg tttccattcc ccctgggcat attgaaaaag ctgtgtggaa 3780

cgtggcggtg ccagacattt ggtggggcca acctgtacac tgactaattc aaataaaagg 3840

gacagatcat aacttcgtat agcatacatt atacgaagtt atccgcgggg atatagatct 3900

ctcgagggggg ggcccggtac ccagcttttg ttccctttag tgagggttaa tttcgagctt 3960

ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 4020

caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 4080

cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 4140

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 4200

ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 4260

ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 4320

agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4380

taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4440

cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4500

tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4560

gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4620

gggctgtgtg cacgaaccccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4680

tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4740

gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4800

cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 4860

aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 4920

tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 4980

ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5040

attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 5100

ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 5160

tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 5220

aactacgata cggggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 5280

acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 5340

aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 5400

agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 5460

ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 5520

agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 5580

tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 5640

tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 5700

attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 5760

taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 5820

aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 5880

caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 5940

gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 6000

cctttttcaa tattattgaa gcatttatca gggttatgt ctcatgagcg gatacatatt 6060

tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc aatttcccc gaaaagtgcc 6120

acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 6180

gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 6240

cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 6300

atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 6360

tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 6420

tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 6480

tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 6540

atttaacgcg aattttaaca aaatattaac gcttacaatt tccatcgcc attcaggctg 6600

cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 6660

gggggatgtg ctg 6673

<210> 3

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gaccaccaag cgaaacatcg 20

<210> 4

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atatcacggg tagccaacgc 20

<210> 5

<211> 61

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cgaagagtaa ccgttgctag gagagaccgt ggctgaatga gactggtgtc gacactagtg 60

g 61

<210> 6

<211> 48

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gatcccacta gtgtcgacac cagtctctaa tttttttttt caaaaaaa 48

<210> 7

<211> 28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgaagagtaa ccgttgctag gagagacc 28

<210> 8

<211> 25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtggctgaat gagactggtg tcgac 25

<210> 9

<211> 37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttaaaggcac agtcaactta gtgtatgtaa acttctg 37

<210> 10

<211> 32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgaaaaacga gttttaatga ctccaactta ag 32

<210> 11

<211> 36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctaactgacc taagacaggg aatttttact aggatt 36

<210> 12

<211> 31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

taaggtgtat gtaaacttcc gacttcaact g 31

<210> 13

<211> 19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

acaccccctc tgatccgcg 19

<210> 14

<211> 24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cggccgctcg tacgtcacta attc 24

<210> 15

<211> 23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tccatgctgt actgtttccc ccc 23

<210> 16

<211> 18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cggaccgact cggatgtc 18

<210> 17

<211> 22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ccgactcagc tggtggtagt aa 22

<210> 18

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ttccctctgt tcccgtcact 20

<210> 19

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tggtgcctca gtttgaatgc 20

<210> 20

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ccgtatcagt gcacgttcga 20

<210> 21

<211> 18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tgggtgcgca agttgaaa 18

<210> 22

<211> 21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

cctcatcaat gcctgcagtt t 21

<210> 23

<211> 24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ctcagtagca gacccttgta agca 24

<210> 24

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

acatcatccc tgcatccact 20

<210> 25

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

agatccacga cggacacatt 20

Claims (10)

1. A polyA gene trapping vector mediated by Sleeping Beauty transposon, characterized in that: the gene trapping vector comprises: SA-EXON-SV40 polyA-TT gene mutation element, SV40 promoter, IRES-SD-ARE sequence 1. Screening marker gene Neo and FRT sequences, the genes are connected in series in the order of SA-EXON-SV40PolyA-TT-FRT-SV40-Neo-FRT-IRES-SD-ARE. 2. An inducible transposase expression vector, characterized in that: the transposase expression vector comprises CMV promoter, rtTA and TRE elements, SB11 transposase, βactin polyA sequence, and the gene is pCMV-rtTA-TRE - Sequential concatenation of SB11-βactinpolyA. 3. A gene insertion mutation system for mouse embryonic stem cells, characterized in that: the gene insertion mutation system comprises: (1) The polyA gene trapping carrier mediated based on Sleeping Beauty SB transposon according to claim 1; and (2) The inducible transposase expression vector according to claim 2. 4. A construction method based on Sleeping Beauty SB transposon-mediated polyA gene trap carrier according to claim 1, characterized in that: the construction method of the gene trap carrier is specifically as follows: pT2/polyA-trap The vector was digested with AatII and SacII, and the SA-EXON-SV40polyA-TT gene mutation element, SV40 promoter, screening marker gene Neo, and IRES-SD-ARE sequence were ligated to obtain pT2/SA-EXON-SV40PolyA-TT -SV40-Neo-IRES-SD-ARE vector, and then clone the FRT sequence to both ends of SV40-Neo by PCR method to obtain the gene trap vector pT2/SA-EXON-SV40PolyA-TT-FRT-SV40-Neo- FRT-IRES-SD-ARE, the nucleic acid sequence of the gene trap carrier is shown in SEQ ID No.1. 5. a construction method of the inducible transposase expression vector described in claim 2, is characterized in that: the construction method of described transposase expression vector is specifically as follows: by AgeI/BsiWI double digestion, TRE - The miniCMV fragment is cloned into the vector pCMV-rtTA-SB11-βactin polyA to obtain the transposase expression vector pCMV-rtTA-TRE-SB11-βactin polyA, the nucleic acid sequence of which is shown in SEQ ID No.2 . 6. A gene insertion mutation method for mouse embryonic stem cells, characterized in that: the method comprises the steps: (a) Construction of the insertion mutation system, including the construction of the polyA gene trap vector mediated by the Sleeping Beauty SB transposon according to claim 1 and the transposase expression vector described in claim 2 for controlling the function of the gene trap vector construction; (b) Genetic transformation of mouse embryonic stem cells and screening of cell clones; (c) Analysis of integration sites of embryonic stem cell insertion mutation clones; (d) Expression analysis of mutant genes. 7. the gene insertion mutagenesis method of mouse embryonic stem cell according to claim 6, it is characterized in that: in the step (b), the genetic transformation of mutant carrier importing embryonic stem cell is specifically electrotransformation, and concrete electrotransformation process is as follows: digest and collect small Mouse ES cells, the number is ¹ ×107 cells, the amount of mutant carrier DNA is 25 μg, electroporation is performed using a Bio-rad electroporator, the parameters are: voltage 250, capacitance 500, resistance ∞, electroporation cup diameter 4mm, wherein: the mutant carrier Including gene trap vector and transposase expression vector, the mass ratio of the two is 1:1. 8. The gene insertion mutation method of mouse embryonic stem cells according to claim 6, characterized in that: insertion mutation clone screening and insertion site analysis, the specific process is as follows: after the cells are electroporated, 1 μg/ml Dox is added to the culture solution , to induce the expression of SB11 transposase. After 24 hours, use 200μg/ml G418 to screen transfected cells, replace the medium once a day, and screen for 1 week. After expanding the surviving mES monoclonal clones, select different cell clones for positive clones Screening and insertion site analysis, the obtained fragments are sequenced, and database comparison analysis is performed to obtain the capture gene site. 9. The application of the gene insertion mutation method described in claims 6 to 8, characterized in that: the method is mainly used to carry out large-scale gene insertion mutation to the genome of mouse embryonic stem cells, and to screen single-cell clones of different gene insertion mutations in batches library, to obtain a cell clone library carrying a single mutant gene and having the ability to differentiate. 10. The application of the gene insertion mutation method according to claim 9, characterized in that: said cell clone carrying a single gene mutation is analyzed for totipotent differentiation ability and the establishment of a single cell clone bank, specifically including cell clone embryonic stem cell marker gene Expression detection and batch screening of single cell clones; Immunofluorescence staining was performed using antibodies to molecular marker genes SSEA-1 and OCT-4, and at the same time, fluorescent quantitative PCR was used to detect molecular marker genes SSEA-1, OCT-4, REX-1, The expression level of NANOG was determined, and alkaline phosphatase staining and chromosome karyotype analysis were carried out to confirm that the cell clones obtained by this mutation method had the totipotency of embryonic stem cells.