CN111440774A - Construction method of IFNGR2 gene melanoma B-16 cell line for stably expressing knock-in EGFP - Google Patents

Abstract

The invention discloses a construction method of an IFNGR2 gene melanoma B-16 cell line stably expressing knock-in EGFP. According to the invention, a green fluorescent protein EGFP gene sequence is knocked into the end of the last exon of an IFNGR2 gene in a cell, so that the EGFP gene sequence and the IFNGR2 gene are subjected to fusion expression. The result shows that the positive correlation between the EGFP fluorescence intensity and the IFNGR2 expression level can indicate the expression level of IFNGR2, and the cell line can be used for the IFNGR2 expression regulation research. The invention provides a powerful tool for finding the expression regulatory factor of IFNGR2 and screening the drug targeting tumor immunity.

Description

Construction method of IFNGR2 gene melanoma B-16 cell line for stably expressing knock-in EGFP

Technical Field

The invention relates to a construction method of an IFNGR2 gene melanoma B-16 cell line stably expressing knock-in EGFP.

Background

Tumor immunotherapy refers to a therapeutic method for targeting and attacking tumor cells by activating the inherent ability of the body's immune system, so as to control the development of tumors and kill tumors. The tumor immunotherapy mainly includes immune vaccine, immune checkpoint inhibitor therapy, adoptive immune cell therapy, cytokine therapy, and the like, in which the immune checkpoint inhibitor therapy is spotlighted with its remarkable clinical efficacy. However, current clinical trial data show that immune checkpoint inhibitors are effective in only about 20% of patients, and the causes and mechanisms leading to treatment failure have not been fully elucidated.

Gao J, et al, found that the lack of IFN γ signaling pathway in tumor cells resulted in drug tolerance of tumor cells, thereby resulting in failure of anti-CT L A4 therapy, Benci J L, et al, found that IFN γ signaling pathway could regulate the sensitivity of tumor cells to drugs at immune checkpoint.

IFN gamma through with cell membrane receptor binding, activation of Janus kinase/signal transduction and transcriptional activator (JAK/STAT) signal pathway, phosphorylated STAT nuclear and then regulation of interferon inducible gene expression. The interferon gamma receptor (IFN γ R) comprises two distinct subunits, IFNGR1 and IFNGR2, respectively. Studies have shown that cell surface IFNGR1 expression is excessive, but that IFNGR2 expression is tightly regulated, the amount of expression determining the responsiveness of the cell to IFN γ signaling. The elucidation of the expression regulation mechanism of IFNGR2 helps to elucidate the regulation mode and mechanism of the IFN gamma signal pathway, thereby providing a new strategy for improving the immunotherapy effect of tumors.

Disclosure of Invention

The invention aims to provide a construction method of an IFNGR2 gene melanoma B-16 cell line stably expressing knock-in EGFP.

In a first aspect, the present invention provides a method for preparing a cell line, comprising the steps of: performing gene editing on a B-16 melanoma cell by using a CRIS-PITCh system, inserting a coding gene of green fluorescent protein EGFP into the seventh exon end of IFNGR2 gene in the B-16 melanoma cell, and performing fusion expression on the coding gene of the green fluorescent protein EGFP and the IFNGR2 gene; the CRIS-PITCh system comprises a sgRNA expression cassette; the target sequence of the sgRNA is (a1) or (a2) as follows:

(a1) position 15884 and 15903 from the 5' end of SEQ ID NO. 1;

(a2) SEQ ID NO.1 at position 15939-15958 from the 5' end.

The CRIS-PITCh system comprises a carrier A and a carrier B; the vector A comprises a Cas9 expression cassette, an sgRNA expression cassette and a PITCh sgRNA expression cassette; the vector B comprises a donor fragment and homologous arms positioned at two ends of the donor fragment; the donor fragment is (b1) or (b2) as follows:

(b1) position 1595 and 2974 from the 5' end of SEQ ID NO. 4;

(b2) SEQ ID NO.5 positions 28 to 1407 from the 5' end.

The homology arms at both ends of the donor fragment described in (b1) are the upstream homology arm shown in position 1567-1586 from the 5 'end of SEQ ID NO.4 and the downstream homology arm shown in position 2975-2994 from the 5' end of SEQ ID NO. 4.

The homology arms at both ends of the donor fragment of (b2) are the upstream homology arm shown in SEQ ID NO.5 at 1-20 from the 5 'end and the downstream homology arm shown in SEQ ID NO.5 at 1408-1427 from the 5' end.

The CRIS-PITCh system can be composed of a circular plasmid (vector A) shown by SEQ ID NO.2 and a circular plasmid (vector B) shown by SEQ ID NO. 4.

The CRIS-PITCh system can also be composed of a circular plasmid (vector A) obtained by replacing 8319-8343 positions from the 5 'end of SEQ ID NO.2 with SEQ ID NO.3 and a circular plasmid (vector B) obtained by replacing 1567-2994 positions from the 5' end of SEQ ID NO.4 with SEQ ID NO. 5.

In a second aspect, the invention provides a cell line produced by the method of the first aspect.

In the examples of the present invention, two cell lines were prepared by the above-described method.

One of these differs from the wild type (B-16 melanoma cells) in that: the DNA molecule shown in the 15886 and 15887 positions from the 5 'end of SEQ ID NO.4 is inserted between the 15886 and 15887 positions from the 5' end of SEQ ID NO. 1. Another difference from the wild type is that: the DNA molecule shown in SEQ ID NO.5 from positions 21 to 1407 of the 5 'end is inserted between positions 15941 and 15942 of the 5' end of SEQ ID NO. 1.

In a third aspect, the invention protects the application of the cell line in the research of IFNGR2 gene function and expression regulation mechanism.

The invention also protects the application of the cell line in the screening of the medicine for targeting tumor immunity.

In a fourth aspect, the invention protects the sgRNA expression cassette or the CRIS-PITCh system described in any of the preceding paragraphs.

In a fifth aspect, the invention protects the use of the sgRNA expression cassette or the CRIS-PITCh system described in any of the above in the preparation of the cell line described above.

The invention also protects the application of the sgRNA expression cassette or the CRIS-PITCh system in researching IFNGR2 gene function and expression regulation mechanism or tumor immunity targeted drug screening.

The invention also protects the application of the sgRNA expression cassette or the CRIS-PITCh system in the preparation of a cell line for researching IFNGR2 gene function and expression regulation mechanism or drug screening of targeted tumor immunity.

Any one of the IFNGR2 genes is a DNA molecule shown in SEQ ID NO. 1.

Any of the IFNGR2 genes is a DNA molecule which has 90 percent of homology with SEQ ID NO.1 and has the same function.

According to the invention, a green fluorescent protein EGFP gene sequence is knocked into the end of the last exon of an IFNGR2 gene in a cell, so that the EGFP gene sequence and the IFNGR2 gene are subjected to fusion expression. The result shows that the positive correlation between the EGFP fluorescence intensity and the IFNGR2 expression level can indicate the expression level of IFNGR2, and the cell line can be used for the IFNGR2 expression regulation research. The invention provides a powerful tool for finding the expression regulatory factor of IFNGR2 and screening the drug targeting tumor immunity.

Drawings

FIG. 1 is a design and flow chart of an IFNGR2 insertion site; (a) the target site of gene editing is located at the end of the last exon, and the donor vector comprises EGFP sequence (b) design of mouse Ifngr2 gene insertion site (c) gene editing flow diagram.

FIG. 2 is a schematic diagram of CRISPR-Cas9 and CRIS-PITCH (v2) vector construction; (a) schematic diagram of CRISPR-Cas9 construction; (b) CRIS-PITCH (v2) construction scheme.

FIG. 3 is a drawing of the identification of IFNGR2-EGFP cell line (a) genomic PCR identification primer design; (b) and (c) identifying the electrophoresis result map by genome PCR, wherein an arrow points to a target band; (d) and (e) product 1 sequencing results plot, blue part is homology arm sequence; (f) flow chart of positive clones, positive cells in box; (g) the fluorescence microscope showed that the positive clones showed green fluorescence.

Figure 4 shows that positive clones were normal for IFN γ reactivity, (a) the relative expression of Cd274 (PD-L1) was up-regulated (b) the relative expression of Psmb9 was up-regulated, n-3, p <0.001, p <0.01, p < 0.05.

FIG. 5 shows that TNF- α and oxaliplatin up-regulate the expression of Ifngr2 and EGFP, (a) and (e) after stimulation of TNF- α and oxaliplatin, green fluorescence right shift oxal: oxaliplatin, (b) and (f) flow observation result statistical graphs, (c), (d), (g), (h), (i) and (j) qPCR analysis results under different stimulations, TNF- α and oxaliplatin stimulation up-regulate the relative expression of Ifngr2 and EGFPMRNA, and IFN gamma cannot up-regulate the relative expression of Ifngr2 and EGFP mRNA.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Statistical analyses in the following examples were performed using GraphPad Prism software, where differences between groups were analyzed using the t-test, and P <0.05 considered statistically significant.

The embodiment of the invention carries out gene editing by using a CRIS-PITCh system, and knocks a green fluorescent protein EGFP gene sequence into the end of the last exon of an IFNGR2 gene in a B-16 melanoma cell, so that the EGFP gene sequence and the IFNGR2 gene are fused and expressed. The experimental design concept and flow are shown in fig. 1.

The IFNGR2 gene is shown in SEQ ID NO. 1. In SEQ ID NO.1, the 15814-15948 th exon from the 5' end is the seventh exon.

Example 1 sgRNA target selection and oligonucleotide strand synthesis thereof

IFNGR2 gene sequence was analyzed, and guideRNA was designed using http:// chopchop. Two sites were selected from several sites and named PAM1 and PAM2, respectively.

The primer sequences were synthesized by Biotechnology engineering, Inc., as shown in Table 1 below.

TABLE 1

In Table 1, underlined sequences are target sequences.

Example 2 construction of Gene editing vectors

Two vectors, CRISPR-Cas9 vector and CRIS-PITCH (V2) vector, are required for microhomologous end-joining gene knock-in. The main structure and construction process of the vector are shown in FIG. 2. Wherein the CRISPR-Cas9 vector comprises a knock-in position sgRNA expression cassette and a PITCh sgRNA expression cassette for making DNA double strand breaks of the genomic and donor vectors. CRIS-PITCH (V2) is a donor vector, comprises a DNA fragment to be inserted into a genome, EGFP and puromycin resistance genes separated by a 2A sequence, homologous sequences at two ends of the fragment are at two sides of an editing position, and an upstream homologous arm and a downstream homologous arm are both about 20 bp.

Construction of CRISPR-Cas9 vector

CRIPSR-Cas9-PAM1 vector at PAM1 site and CRIPSR-Cas9-PAM2 vector at PAM2 site were constructed by referring to the following steps, respectively.

1. The upstream and downstream primers in Table 1 were annealed to form double-stranded DNA molecules.

2. After digestion of the pX330A-1X2 plasmid (purchased from Addgene) with BbsI, the cleavage product (about 8941bp) was recovered from the gel.

3. And (3) connecting the double-stranded DNA molecule prepared in the step (1) with the plasmid enzyme digestion product prepared in the step (2) by adopting T4 DNA ligase to obtain a pX330-1x2-sgRNA recombinant plasmid.

4. The pX330-1x2-sgRNA recombinant plasmid was digested with BsaI, and the digestion product (about 8471bp) was recovered from the gel.

5. After digestion of the pX330S-2-PITCh plasmid (purchased from Addgene) with BsaI, the digestion product (ca.443 bp) was recovered from the gel.

6. And (3) connecting the enzyme-cut product prepared in the step (4) with the enzyme-cut product prepared in the step (5) by using T4 DNA ligase to obtain a CRISPR-Cas9 vector.

The CRIPSR-Cas9-PAM1 vector is a circular plasmid shown as SEQ ID NO. 2.

The CRIPSR-Cas9-PAM2 is a circular plasmid obtained by replacing the 8319-8343 position from the 5' end of SEQ ID NO.2 with the SEQ ID NO. 3.

Construction of the second, CRIS-PITCH (V2) vector

1. Primers respectively containing sequences at the upstream and downstream of the PITCh gRNA, IFNGR2-PAM1 and IFNGR2-PAM2 sites are designed, and the specific details are shown in Table 2.

TABLE 2

2. PCR amplification was performed using a PCR amplification primer using a PCRIS-PITCH (V2) -FB L vector (purchased from Addgene) as a template, and the amplification products (about 1500bp EGFP-2A-Puro fragment containing the homology arm of the target gene and 4500bp vector backbone fragment) were separated and purified by agarose gel electrophoresis.

3. And (3) recombining and connecting the purified product obtained in the step (2) by using ExnaseII recombinase to obtain CRIS-PITCh (v2) -PAM1 and CRIS-PITCh (v2) -PAM2 vectors.

The CRIS-PITCh (v2) -PAM1 vector is a circular plasmid shown in SEQ ID NO. 4. In SEQ ID NO.4, the position 1567-1586 from the 5' end is an upstream homology arm, the position 1595-2974 is a donor fragment (wherein the position 1595-2311 is an EGFP gene sequence, the position 2312-2374 is a T2A spacer sequence, the position 2375-2974 is a Puro sequence), and the position 2975-2994 is a downstream homology arm.

The CRIS-PITCh (v2) -PAM2 vector is a circular plasmid obtained by replacing position 1567-2994 from the 5' end of SEQ ID NO.4 with SEQ ID NO. 5. In SEQ ID NO.5, the upstream homology arm is located at positions 1-20 from the 5' end, the donor fragment is located at positions 28-1407 (wherein positions 28-744 are the EGFP gene sequence, positions 745-807 are the T2A spacer sequence, positions 808-1407 are the Puro sequence), and positions 1408-1427 are the downstream homology arm.

Example 3 establishment of melanoma B-16 cell line stably expressing IFNGR2 Gene knock-in EGFP

First, establishment of cell line

1. The B-16 melanoma cell line (ATCC, accession No.:

CRL-6475^TM) Inoculating into 6-well plate, culturing in 1640 medium containing 10% fetal calf serum at 37 deg.C and 5% CO₂Culturing under the condition until the cell density reaches 60-80%.

2. After completion of step 1, transfection procedures were performed using plasmid transfection reagents L ipofectamine (TM) L TX (Invitrogen) according to the instructions, and plasmids of group 1(CRIPSR-Cas9-PAM1 vector and CRIS-PITCh (v2) -PAM1 vector) and group 2(CRIPSR-Cas9-PAM1 vector and CRIS-PITCh (v2) -PAM1 vector, in a ratio of 2:1) were transfected into cells, respectively, and a group transfected with pmCherry-C1 plasmid (purchased from Addge) was set as a positive control.

The procedure was to mix the plasmid and transfection reagent in 150. mu.l: standing for 15min after 6 mul is uniformly mixed, then dropwise adding the mixture into cells, absorbing and removing culture solution after 6h of transfection, adding fresh culture solution and continuously culturing overnight, and observing the fluorescence condition of a positive control group under a fluorescence microscope after 24h of transfection to confirm the transfection effect; cells were transferred from six-well plates to 10cm dishes 72h after transfection, the medium was replaced with puromycin (2mg/ml) containing medium and the medium was changed every three days until a monoclonal cell mass grew in the dish. And (3) picking the monoclonal cell clusters to a 96-well plate under a microscope for continuous culture, transferring the cells to a 6-well plate for continuous culture after the cells grow full, and performing subsequent identification.

Cells were first identified by PCR, and the primers used for PCR are shown in Table 3.

In Table 3, primer pair 1 and primer pair 2 were used to identify positive clones of group 1 (i.e., transformed into CRIPSR-Cas9-PAM1 vector); primer pair 1 and primer pair 3 were used to identify positive clones of group 2 (i.e., transformed into CRIPSR-Cas9-PAM2 vector). A schematic of the amplified region of the primer pair is shown in FIG. 3 a.

Amplification using primer pair 1 should yield product 1 with a molecular weight of about 500 bp.

Amplification using primer pair 2 should yield product 2 of approximately 1450bp in size.

Amplification using primer pair 3 should yield a product 3 of approximately 1450bp in size.

TABLE 3

Genomic DNA of the cells was extracted and PCR amplified using the primers shown in Table 2, respectively.

The PCR results of one positive clone E10 selected from group 1 (i.e., transformed into CRIPSR-Cas9-PAM1 vector) and one positive clone B4 selected from group 2 (i.e., transformed into CRIPSR-Cas9-PAM2 vector) are shown in FIG. 3B and FIG. 3 c.

The product 1 of both clones was purified and sequenced, and the alignment results are shown in fig. 3d and fig. 3e, indicating successful insertion of the EGFP sequence into the genomic target site.

The positive clone E10 was sequenced and distinguished from the wild type by: the DNA molecule shown in the 15886 and 15887 positions from the 5 'end of SEQ ID NO.4 is inserted between the 15886 and 15887 positions from the 5' end of SEQ ID NO. 1. The positive clone B4 differs from the wild type in that: the DNA molecule shown in SEQ ID NO.5 from positions 21 to 1407 of the 5 'end is inserted between positions 15941 and 15942 of the 5' end of SEQ ID NO. 1.

Flow analysis was performed for E10 and B4, respectively, and fluorescence expression intensity of EGFP was observed using a living cell imaging system (DV). The B-16 melanoma cell line was used as a control.

The flow results are shown in fig. 3f, the green fluorescence intensity of the positive cell line is significantly higher than that of the control group, and the positive rate reaches more than 95%. The fluorescence observation results are shown in fig. 3g, and green fluorescence can be successfully observed in the positive cell line, but cannot be observed in the control group, indicating that the inserted EGFP sequence is successfully expressed.

30 clones are respectively picked from PAM1 and PAM2 for identification, 7 positive clones are identified by PAM1, the positive rate is about 23.3 percent, 1 positive clone is identified by PAM2, and the positive rate is 3.3 percent.

Example 4 detection of cell lines

1. Clones E10 and B4 prepared in the examples were inoculated in 6-well plates using 1640 medium containing 10% fetal bovine serum at 37 ℃ with 5% CO₂Culturing under the condition until the cell density reaches 60-80%.

2. After completion of step 1, samples were collected simultaneously with the solvent control group for the same time after 48H of treatment with IFN γ (R & D485-MI-100) (20ng/ml), 48H of treatment with Oxaliplatin (Oxaliplatin for injection, Nanjing pharmaceutical factory Co., Ltd., national drug Standard H20000686) (2. mu. mol/ml) and 3H of treatment with TNF- α (R & D Systems, 410-MT-010/CF) (20ng/ml), respectively.

3. Extracting the total RNA of the cells collected in the step 2 and performing reverse transcription to obtain cDNA. The cDNA was used as a template for real-time quantitative PCR detection, and the primers for detection are shown in Table 6. Each group was repeated 3 times, and relative expression amounts and fold differences were calculated using GAPDH as an internal reference gene and subjected to statistical analysis.

TABLE 6

To determine whether the B16-IFNGR2I-EGFP cell line can respond to the stimulation of IFN gamma, IFN gamma treatment is carried out on positive clones, and the expression of downstream target genes Cd274(PD L1) and Psmb9 of the IFN gamma is detected, the result is shown in figure 4, the result shows that the expression of Cd274(PD L1) and Psmb9 is remarkably up-regulated 48 hours after the stimulation of the IFN gamma (20ng/ml), and the up-regulated amplitude is equivalent to that of the parent cell B-16, which indicates that the two cells can respond to the stimulation of the IFN gamma, and indicates that the IFN gamma signal channel in the cells is complete.

Flow cytometry analysis was performed on cell clones E10 and B4 after stimulation with TNF- α and oxalliplatin, respectively, and RNA was simultaneously extracted for qPCR analysis, flow results are shown in fig. 5a and 5E, and after treatment with TNF- α and oxalliplatin, the green fluorescence peaks in positive clones E10 and B4 cell lines shifted to the right, indicating increased EGFP expression, the mean fluorescence intensity values of the main cell population were calculated and statistically analyzed, showing that TNF- α and oxalliplatin treatment significantly enhanced fluorescence of E10 and B4 cells (fig. 5B, f), qPCR analysis shows that TNF- α and oxalliplatin treatment significantly increased both IRNGR2 and EGFP mRNA levels in E10 and B4 cells at the same time, the above results show that the intensity of green fluorescence in B16-IRNGR2-GFP cells can reflect the expression level of IRNGR2 in cells.

Sequence listing

<110> biomedical analysis center of military medical research institute of military science institute

<120> construction method of IFNGR2 gene melanoma B-16 cell line stably expressing knock-in EGFP

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gcacggactg gaatccagtg cattctgtca ggcagtttgg tcccagggac acaaaagtaa 4620

caggggtctt tagaaccatc cggagaaggt cagcacccct gccctggcct gtgacacgag 4680

acaaaaccac aggccaaaca caaagcaagg aagagagcgc ctgggccttg agtgggttct 4740

gttccatttt tttttttcat ggttaacagc agtctgtcag cgcaatgttc gttctccgca 4800

tcttctgagc ctgctgttcc aggcccagag agaaagcacc cctgaatccc gcagcacctc 4860

agcttctcaa agtgtctgcg tggcgctgaa gagtaactga tgggcagctg tgggaaaaaa 4920

aatgtgagct gagcctacac agcacacaaa gtcgctcctt ccacagaaag actgtgttga 4980

ggacggacgc actgtgactg gtctccagac ccaaatacct tatccagagg caagcacaaa 5040

gacagtagca gaggagcctc tcgaaggact ccagtgtgaa tcacactctg gaatacaatc 5100

gagcaagaca gtagaggtga gagcaggatt atacgtttac cccagggctc agtctgagtg 5160

acagcaggga cgctccctgg agagggagct gcagagcaag gtcccaagta cggctgtgct 5220

gtttcccagg agcagagagg gtgaggttcc aggacgggcc atccagaagt gctttatggt 5280

gtgcggacag tctctggcac ttcccgagga gtccacagtt ctgagactca ttttcttcag 5340

tgaagaagat tagataaggt tcccaagaaa ctcccggtgg acagccatgc tgtgtgaagc 5400

tttccttata gccagtctga gaactccgtc ttgccaggtc aacttttttt tttttttttt 5460

tttttttttg agacagggtt tctctgtgta gccctggctg tcctggaact cactctgtag 5520

accaggctgg cctcgaactc agaaacccgc cttcctctgc ctcccaagtg ctgggattaa 5580

aggcgtgcgc caccatgccc agcccaggtc agctttttgt tgtttgtttt taagtgttgg 5640

gcatccaacc tagggctttg agccatgagg cgcgtggtgc gcaactgagt tccactccca 5700

cccccacttc cagccctgaa cgtgcgcatg cgcgtaaagc aaactaaaac tcccaaggta 5760

tggttgaggg gaagatctga gggagagtac agccggaggc atccggagga gtccggagca 5820

gggaaagtag tagatggaca tggctagcag actggaccag gccaagaaga gaggagatag 5880

agagtgagga agacacaaga ggcccaagaa agcgcatagc ccaaatgtca cgactaccta 5940

cctacctacc aggatgagaa gctgggggcc agaaatcact ggaagccaat gagctggagg 6000

tgaggtgaag tttaggatcg ggtgagctga gctgagggga gctacaggtc ctgagtgggc 6060

ctgagtgggc ctggaggcca cagacacttg gtatgctaat aggcaccaca gttaagccat 6120

ttgtcctgag tctcttttgt atctgagaac cctgatagct ttcctcttcc acatgtggtt 6180

aggttctttc taacctggtc ccttggcgtc cctctcttaa atggatgttt agattgtgag 6240

aataggaacc cagtggagga agagaatatt ctttctggtt cctccggaga tgtgagagct 6300

ggtgagagga agatgtgtta ggcagaataa tgtcctttaa acgatgtcca ggcactgatc 6360

cccggacacc ttagggatat gtgatctgac agaaggggct ttgctggtat gactgagttg 6420

aagatcctga cggggagctg actggattct accctacgtg gactcacggt cattaacagg 6480

gtcctggaaa gatagagtaa gaaagagaac cagagggagg gtaacacaag gctgactggg 6540

cccagcattg acagagaaaa gggccacaag ccaaagaatg ccaatagctt ccagagcaca 6600

gaaaagggag atgccctggc gactccagaa ggaacaagcc ccacccccgc taccatctcc 6660

accctaggcc tgcttcacac acctgacctc aaagggatga taagtgataa atgtcgtgaa 6720

ataattgaga tgatatgtct ccgttggtaa aagctcttgc gtttggggga tgtgctacag 6780

cgctggcaga aactcaatgt gggcaggaaa cgctgctgtc tctgagattt gttgatgggt 6840

gcctgacttc cctggcttct gccaaaccca cagcaagctg accgcgcctg ccctccgcct 6900

tgctgctttg cccccctccc cgtcccgttc ccctgaggcc tgtgtgaatg aatggtctgc 6960

agtggggggg gggggggtgc tggaggcgcc tccgcaccct tcttctgact tggaggggct 7020

ctctgaaaac gtggtttggt ttgtgtttcc tgctttgctt tctggagccg catgtcttcc 7080

ttcccacctc tgaatgtttc tcgttaatct tcccatcaga gaaagtgtta cgacgatttc 7140

tcttttgagg gaaggttcca atgttttcag tctctagccc agcccacccg atatctgcct 7200

gtggcgatgg ttggaactgg tctcattggg gaatctgcta cccccacccc cacccccggg 7260

aatgtgttct tttgtgattt gtggctctca ggggtgggag acaagttaaa attgaacatt 7320

tcctcagtgg cttcgaacct gggatgcaga aagttatccg gatcccagcg atggaatgct 7380

gagagttttt gaggagcaaa tgatgtctga tcttgcccac atagcaaggg cctttgggag 7440

ttgactgact gattttgggc atctgcatga acacatacat gcagggtcat cttggtactc 7500

tctctgggct tagcaggtgc tcactctatg gggtggtctg ttattggtta ggattagggt 7560

tattcctgta gaaagatgga atcttgggca gctcacagtc aagtgccaca tatgatagat 7620

gggggaaccc agagtagtct agcctgtggt ccccacgacc tagagggtga cctaaaaaaa 7680

aatgtctccc atagtgccct cctcacaatc aggcccagga gggggtgagt ctgggagatc 7740

agctgctctt gttccagagc caaactgcag ccagagctgt gttgaataaa tgactttgac 7800

aacaggcacg gaaacattgt tgtaattctc tcaatttttc cccctctcct ctgccccttt 7860

tcctcctctt gttccctgtt cctcccaaga ctcgttttcc cagcttgcgg cccctctgaa 7920

cccaaggctt cacctgtaca atgatgagca gattctaact tgggagccgt caccttccag 7980

caatgaccca agaccagtgg tctaccaggt ggaatatagc ttgtaagtct gatgctctgc 8040

gggtgtcctt gatgctctgc aggagtcctt gatgttcagt gagtgtcctt gctgggtgag 8100

acctgttggg gaagttggct gggagcctgc agtgagatcc tgaccccggg ctttcagcat 8160

gactatggtg agacccccta tgcacacaaa agcttcaagg acagagatct catcacctcc 8220

tgctcatctg aggcgaactt aagcctggct gttaaagagc tgtggctgct gggacctcct 8280

ttattggtga cacatgatcc ctttgctctg tacagagtga gggttctctt ctgccctgga 8340

aaggcaagag atagctttca ggtttataac ctcaaagtcc aaactgagcc tactgaggaa 8400

tgtgttcagc agactacagt gaactctcag tttctcacct ggttttctgt tctgaataat 8460

ctagtgttca cgccctttgt gctgcctctc gcctcagtcg caggccacca cttgatttgc 8520

aaagccgtat tcactaagaa actgctcagt caaacggttt tgttttgttt tgttttgttt 8580

tcttggtcac atatgttagt tgaagatcaa gctgaaagca ctgggcacac ggttataaac 8640

aaaaactgat tgttttgaga tgagcattcc tgtttggctc agatagctct ggagctagag 8700

atcctcctgc ttcggcctcc aggaggagct gagactcctg ccagcccgcc agtgtcactg 8760

cactctgagc tttgtttaaa agacttccac agagctttgt attacagact cacttctttg 8820

actctagggt tggttgtttg tggggtgggt gggtgtgtgc acatgtgtgc gcaagcctct 8880

ctcacacaca tacacacatg cacgttcaca tacgcacaca gctgtgtgga agccagggag 8940

catggcaaag tgccttcctt agtcccgctc cccgtatttt tggggacggt ctctcactga 9000

acccagagct ctccaattca gctagattgg cagaccaggg aatcatcggg tttctcctct 9060

ccacgcctgc cccacccctc tccacgcctg ccccacccct ctccacgcct gccccacccc 9120

tctccacacc tgaacgctgc cggggtcact ggggttttgc aggtttgctt ggatgctgag 9180

ggtctgagcc caggctgaca gtttaacagt agccatttcc ccagctccct tgctttcttt 9240

cttcttagta aacattttgt atctttttta tcaggggttg aggttggttt tagtaagaaa 9300

agcattggta aacactaaag caggctggtc ccccaccctc actccctctg ctgcagagct 9360

ccgctcttgt gaggggaaga ggtctctggt tttgtttgac tttaaaaatc agttttctta 9420

tttgaggttt tttttttttt taactgattt tgagactaag atttaaaaac tgtgtttgtc 9480

tgtggtcaag catgactgcc attgtaaagg aaggcgtccc cttagctttt tatgaaggtc 9540

acagtagcaa ggccaaatct ataggagagt ctaggttcgt gtcaggttag cagttaaaat 9600

ccaccatctc ggatggccag cgtgctctcg aacgcgcaac atattaggta acgtctgaaa 9660

gcacaagtcc agagtgaccc tgtgcaaagt gtagagagaa tctcgtctct tgcactgtgt 9720

ggaagcctca ccgtgtgcca agaaaaagcc gactgccttt tagcaagagg caggaagaaa 9780

tatgtggggt ggaagagggt agaactctgg gaattcatca gaggcactgg agataggagc 9840

cgtaaccagc cagccatgtg gccttcagaa ttgaataacc gggaactaaa caaacaggaa 9900

cgagctaaag cttgtggcct gggcatttag tcataattaa gtctccggac ggaaaagctg 9960

tggctacagc ctggaagccg ggtgactctc ctgattagta aaccttagac cgagttggtg 10020

gcgctcgatg aactgtacga ttgagccgtg ttcatttcat tgctcatcta aatgcagcca 10080

gaatcatctt atttttaccc tctgtacgca aagggactcc ttccctcaga acccccgtcc 10140

cataaatctg tcttacatga gggcacatag aaataaaaca cagaatttta aagttttaga 10200

ggaagaaaaa aaacccaacc cattcattct gtgtcttgaa tgtcacagag gaagtcacag 10260

agaccagggg ttcatgggtc tccatgtggg atggttccgt tccggctgag ggagcctgaa 10320

cctcagtcac tgccgctgac tgaggcttcc gggtgaagag aggtggagga gaatcactat 10380

taatggatac cagatggggg tttggagggg agggctaagt aagaacatat tttaaaaatc 10440

agtcacagcc aagcagtggt ggcgcatgcc tttaatccca gcactcggga ggcagaggca 10500

ggtggatttc tgagttcgag accagcctgg tctacagagt gagttccagg acagccagga 10560

ctacacagag aaaccctgtc tcagaaaaaa aaaaaaaaaa aatcggtcat actggtggtc 10620

tcctaacctt gtgcacaccc tgagagcatt ggatacttga gtaggcgcag cgtatgacct 10680

aatctagcca agagtttctg tatgtgacct ttgcccaaaa gatgaatgag taagcgggtc 10740

ctcgggagga gttaatgtct ctagagaaca gggttactcg cgaagagaga attctgtgag 10800

ctgagatctt gttttccctc ccagcatcga tggctcttgg cataggttgc tggagccgaa 10860

ctgtacggac atcacagaga caaagtgtga cttaacagga ggcggccgct tgaagctttt 10920

cccacaccca ttcacagtct tcctgtgvcg ggtgcgagcc aagcgaggga acctcacttc 10980

caagtgggtg gggctggagc catttcaaca ctatgagaat ggtaagagaa cgtgggaaga 11040

aagctttaca ggggccagag gggcaactca gccgtgcaga gcactctggc tcttgcagag 11100

gaccggagtt cagttcccag cacccacatt ggggtgctcc caactgtctg agactccagc 11160

tccaggcatc taaacgccct tctctggcct cctcacgcac ccacactcac acgcagataa 11220

ataaaataga cacaccttta aaaagaaatg tcagtcccag gttttctcta cgtgcttctt 11280

cattgatcag tacggaaccc accctaggcc ttggtcctgg accccatcct aatacaccag 11340

agacaaatgg tgaggttgtc agagaccact gacctcaggc tgatgctaaa gaatagggtt 11400

tgctgcacca ggctctgggc tctaaatctg accccacgcc agttacttta aacagtgttt 11460

aatgggaagg tagttacagc catatttttg tttcctttgc tatggtgctg gggagcaggg 11520

acctgatacc tccctcctag gcaggtgctg tactgctgag cccccatatg catgcgtgca 11580

tgcgttcgtg tgtgcgtgcg tgcatgtgcg cgcacacaca cacacacaca cacacacaca 11640

cacacatgct caaaattcaa gtataagaca acagatgtga ccgctgtcta catagtcaat 11700

tctgtagtaa gcagttagtg tttttggctg tgtttttttg tttgtttttt gttttttaag 11760

aagtataatc ctagccgggc gtggtggcgc acgcctttaa tcccagcatt tgggaggcag 11820

aggcaggtgg atctttgagt tcaaggccag cctggtctac agagtgagtt ccaggacagc 11880

cagggctaca cagagaaacc ctgtctcaaa aacaaacaaa caaacaaaca aacaaacaaa 11940

cgaagtataa tcctaggtag catggcagat tttattgtta tgtgttgtta tcacaacagc 12000

aaaatgtgat agaaaattta aaataaagct ggataaatta ataaataaat aaaagatccc 12060

ccaaataacc cagagccatt aaaatgccag cttatttaaa agtgagctgg gggataaggg 12120

tggcacacac ctttagttgc agaacttggg aggttgagac aggcagatgt ccatgagttc 12180

aaagccagcc tggtctacag agtgagttcc aagcaagaca gagccacaca ttgagagacc 12240

ttgtctcaaa aaatcaactg tagaggaaaa ctcaatgatg cggtaaatgt tatatcacac 12300

tgtcacttag tcctgtttac ctacctgtaa catgtctcgt gttccacgtg gacgtggtag 12360

actgggatcc tcgtatgagg cagagcaggc ggcgtctttt tccctcactt aacatcatac 12420

tctccagagc cacacacttc tgtgcaaatt tcaaaccttg cacatctttt ctaccctgaa 12480

tgaaaacttc gcctccagtt taagtttctg ttcttgtctc tctgggattc tgacgcctgc 12540

tgggattcta caagctttgc ttgcagtcaa acaactccca gctgagagac agcatctcag 12600

ctagaacaat cttgaatgcc ttgagcaaga ccaaaaaccc ttgcagagtg ctgcacggcc 12660

ctccctgatg ggaacggaaa caacttccat gtagccctga tagatcccgg ctcacagggc 12720

ctttcagcag ggccagtcct ggatgctagc tccctggtaa acctcctgtt cagaacgggc 12780

atctgagtgc agcccattgc gagtgtaagt gtgaacgtgt gtgtacacac agtgtacata 12840

cgtgtggtgt ttaataggca cacacagtgt atacgggtgc ctgtgtcact cacgtgaggt 12900

ctctgtgatt ctatttcagt tactgttgga cctccgaaaa acatctcggt gaccccagga 12960

aaaggttccc tcgtcataca cttctcccct ccctttgatg tgttccacgg ggcaactttt 13020

cagtatcttg tccactactg ggaaaagtca gaaacccaac aggaacaggt aaggcccctt 13080

ttcttctttg tttatggttt ttttctcttt agagtttttt tttttttttc attaagcctc 13140

atgaggtgtc cagaacgtca caaatcttaa tttctagtat agaaatttct cctgtaaaac 13200

taaagcctcg ggctggggag atggttcggt gggcacagcg tttgctatgt gatcccaaga 13260

agctgagttc actccctccg cacctctgta aagtccggca tgttggtgtt ctatctctta 13320

tctccgttat ttacagcccc tcagtcttgt taccatttgg ttgtgttcac tgtggatttg 13380

aggagtcact agaaaagcta gaagctgcat ggccccatct gtccctctgc ttgtctgtct 13440

gtctgtctgt ccgtctgacc tgtgtggctc tggtgctgtc tacacaggtt gaaggccctt 13500

tcaagagcaa ctccattgtg ctgggcaatc tgaagccata cagagtatat tgtttacaaa 13560

ctgaggcaca actgattttg aaaaacaaaa aaatccgacc acatgggctc ttgagcaatg 13620

tatcctgtca cgaaacaaca gcaaatggta ggacatacct tcatgtatcc attacctgct 13680

gcaaaaaagg cttctcctga agtggttagg atggcatgct gtggcatgtg gctggtggtc 13740

ggagaagaga ccggaagtgc ttacccgaca ggagaattat attgaggtct cagcagcaat 13800

ggctgtgtga ctaggctgag cagcagccac aacgcctggg accaggagta tgtcagattt 13860

gtggtcgtct tgtttcagat tttaaaatgt ttgcatatat atataaaatg aggtgtctta 13920

gggatgaatc ctaaatctga ccataaaatt cccttatgtt ttcatagaca ccttgcatgc 13980

atagcctgaa ggaggcttta tataatattt ttagtacact ggtatagaat tttttattgc 14040

attttttgct tacttaagtg gggggtttgt gtgcatgtgt gtgtgtgtgt gtgtgtgtgt 14100

gcacgcgtgt gtgtgggagc gtgcatgcat acatgtcatg tgagtacact tgagtggccg 14160

tcagaggaca ctctgtggta ttgtgacagg atagaggacg ttcacactgc cctctgctgt 14220

cacacatggg tcttcaggac aaaatgcagg cctgtcggtc ttagaggcag gcaacttcat 14280

ttgctgaacc atctcaccgc tccaattgca ccggggccca tcaggtgaat ttggatgtgg 14340

aattttccat ttgtggtatt atgacagggc agtttttgat ttttgaatag aaattttcag 14400

gttttacatg ttcaggtcaa aattattagt ttaagcgcat attgaggcaa gggggtagag 14460

agggcctgtg ggtcttcctg ggacgagaaa acggcccagc agtgggagat ggggcgtgga 14520

ctgctgctgg cggcttgctc acttctgtgt catcttcttt agcctccgcc aggctgcagc 14580

aagtcatcct gattccgttg ggcatcttcg cattgctgct cggcctgacg ggcgcctgct 14640

tcaccctgtt cctcaaatac caaagccgag tgaagtactg gtttcaggct ccgccaaaca 14700

tcccggaaca aatcgaagag gtcagtgcgc agatgcgcga gtgacccgct gtggccccgc 14760

cccctcgtgg taggcgagaa tggacacccc gtgccgtcct ctgccccccc cgcccccgtg 14820

cgtgggcaca tgcccaccca catacgttca cacagaataa ctaagtagta aaatggctgt 14880

tgggttaggt cagagtctat ataacacgtg gctcgtggtg atgggttaaa ggtttccctt 14940

gttgacactg ccaaagagta acagttcccg atggcccttg agctttcact gggctcgtcc 15000

tgactctcag gtacagccct cagaggaact gagcctgggc caccacagtt gtttctctgg 15060

ggcagaaatc taaggttttg ctgtactttc ttgggaaagt gtgaagaact gccagaggct 15120

gctgacgtta cttgagagta gagattggtt tccgcttctg tgtctgaggt ttcacatggc 15180

acgcacatga cttctgttcc tctttccatt ggccgatagc acttctaaga gctgcggatg 15240

gagccctgga atcctggggg gtttatagcc aagagcaaaa gggaaagaaa gttcagggct 15300

gtgagacccc tcttcactgt gaggatccta gccccaccca gatgcgtcca gggttgtgtg 15360

caggtggaag gaggggctag agtttaggga cgttccctgc tttatcctgc cacagctggg 15420

cacagtgggg attcccactt ttcctgagtt ctgcccttcc cagctttcat cagatgaaca 15480

gaggatggag cccaggccct tgtactttct agacaagcgc tcttgctctg agctacacct 15540

gggcctgcgc cattacagag atgagggctg aagaggcagg cacgtgctgg aaatcagggc 15600

tgagcatgag ttagcagcag tgtgccctta ccgtgtgcca ggcctgggct tgacccctag 15660

ggtcacaaca gttaacaatg acgattggtg gtggtatcac ttcattgatg gacaaagtgg 15720

agttgtacct gattataata tgactacatc aactacaagt agggacattg tatcggactg 15780

tctgtaactg gctggccttc tatccttttc tagtatctaa aggacccaga ccaattcatc 15840

ttagaggtct tggacaagga cggttcaccg aaggaggact cctgggactc cgtgtcaatt 15900

atttcttctc cagaaaagga gcgagatgat gtgctccaaa caccgtgaac caggccaggg 15960

tctctgcttg cccaggaggg cagcgatcag tgcacccgag agagatcccc agggccccag 16020

gactggggaa gatggtgtag tgtttgttct ttatgagttt tctggatgct acaagtattt 16080

aaaaggattc cacagaaaga tatctcagaa acaaaacaaa aacaaaaaca aaccaagcgc 16140

ttctcttaaa cactaaaaag acaactgctc attactggca acgtggcggc aaacctgtgt 16200

gtggtgcttt ctccagtgtt agctgcccct gctgtcagaa gactgggtcc ttccttgcct 16260

tcactctgga aaatgagttg tcccagccaa ccctgcctgt cacaaagagg ggcccttagt 16320

gtccccttca tgtccaatgt gatggcttaa atgtgagacc cctgggctcg gtgcttgcct 16380

agcacgtaca cgccccaccc accccagcac ctcactcaca gagaggtgat taatcttgta 16440

aataatgtcc tggtaattta agtctttttt tttttttttt aatctagaga taaaagattg 16500

tatgttaaac tttttttttt tttaagtcta tctgccgggt ggtggtggtg cacaccttta 16560

ctcctggcac ttgggaggca gaggcaggag gatctctgtg agttcgaggc cagcctggtc 16620

tagagtcctg gtctaggaca gccaaggcta cacagagaaa ccctgtctgg taaacaaaac 16680

aggaaaaaga agtgcacgtg tttcttcttt tcttgtccct taccacgtcg ccttctttgc 16740

ctgttttccg taaagtattg acttgtgaaa agtcttagcc aggcatggtg acacatgcct 16800

ttattcccag cacttgaaag cagaggccag tagatttctg tgagtttgag gccagcctgg 16860

tctacatagt gagttccagg acagccaggg ctatgtagag agatcctggc tctctaaata 16920

aataataaat aaataccaaa cttaaaaata 16950

<210>2

<211>8951

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 60

gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 120

atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 180

ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat 240

gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg 300

tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 360

atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg 420

cgccaggcgg ggcggggcgg ggcggggggc ggggcggggc gaggcggaga ggtgcggcgg 480

cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc 540

ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg 600

tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc 660

cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt 720

aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc 780

ctgaaatcac tttttttcag gttggaccgg tgccaccatg gactataagg accacgacgg 840

agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa 900

gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct 960

ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag 1020

caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg 1080

agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag 1140

aagaagatac accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga 1200

gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga 1260

ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca 1320

cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc 1380

cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct 1440

gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt 1500

gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa 1560

ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct 1620

gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac 1680

ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga 1740

cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct 1800

gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa 1860

caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca 1920

ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga 1980

gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca 2040

ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact 2100

gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag 2160

catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt 2220

ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc 2280

ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag 2340

cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca 2400

gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc 2460

caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata 2520

cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt 2580

ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt 2640

caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc 2700

ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa 2760

tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag 2820

agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa 2880

gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg 2940

catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc 3000

caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca 3060

gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg 3120

cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa 3180

agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac 3240

cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa 3300

agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga 3360

gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga 3420

catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga 3480

cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa 3540

cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc 3600

caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag 3660

cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa 3720

gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct 3780

gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga 3840

tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct 3900

gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt3960

gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat 4020

cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga 4080

gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac 4140

cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat 4200

gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc 4260

tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa 4320

gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt 4380

ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat 4440

ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga 4500

agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg 4560

ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc 4620

ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc 4680

cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat 4740

catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa 4800

agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat 4860

catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac 4920

caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca 4980

ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa 5040

aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagtaag aattcctaga 5100

gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 5160

cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 5220

gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 5280

gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa 5340

cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg 5400

cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg 5460

cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 5520

tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg 5580

cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc 5640

ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 5700

atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 5760

ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 5820

tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 5880

actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt 5940

taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta 6000

caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 6060

gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 6120

acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 6180

cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 6240

taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 6300

tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 6360

aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 6420

ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 6480

aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 6540

acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 6600

ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 6660

gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 6720

atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 6780

acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 6840

tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 6900

ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 6960

aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 7020

aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 7080

ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag 7140

atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 7200

aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 7260

accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 7320

tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 7380

tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 7440

tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 7500

cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 7560

caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 7620

cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 7680

cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 7740

gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 7800

acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 7860

atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 7920

cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 7980

gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 8040

tcctggcctt ttgctggcct tttgctcaca tgtgagggcc tatttcccat gattccttca 8100

tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac 8160

acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca 8220

gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc 8280

gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgaataatt gacacggagt 8340

cccgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 8400

agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc 8460

tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctctagag gcatgtgagg 8520

gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa 8580

ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt 8640

aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc 8700

ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa 8760

acaccgcatc gtacgcgtac gtgttgtttt agagctagaa atagcaagtt aaaataaggc 8820

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt ttagagctag 8880

aaatagcaag ttaaaataag gctasgtccg tttttagcgc gtgcgccaat tctgcagaca 8940

aatggctcta g 8951

<210>3

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

caccgctggc ctggttcacg gtgtt 25

<210>4

<211>6032

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc 60

ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 120

tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 180

ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 240

tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc 300

tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 360

actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 420

cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 480

gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 540

tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 600

ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 660

ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 720

cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 780

cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 840

gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc 900

tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt 960

cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg 1020

taaaaacccg cttcggcggg tttttttatg gggagagttt agggaaagag catttgtcag 1080

aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg 1140

agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata 1200

attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa 1260

agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa 1320

attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt 1380

attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc 1440

ttcatgcaca gtggcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg 1500

ggtcattagt tcatagccca tatatggagt tccgcgttac atagcatcgt acgcgtacgt 1560

gtttggaccg aaggaggact cctgggacgg atccatggtg agcaagggcg aggagctgtt 1620

caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 1680

cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg 1740

caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt 1800

gcagtgcttc agccgttacc ccgaccacat gaagcagcac gacttcttca agtccgccat 1860

gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac 1920

ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 1980

cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 2040

caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg 2100

ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 2160

cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag 2220

caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 2280

gatcactctc ggcatggacg agctgtacaa gggaagcgga gagggcagag gaagtctgct 2340

aacatgcggt gacgtcgagg agaatcctgg acctatgacc gagtacaagc ccacggtgcg 2400

cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg cgttcgccga 2460

ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg tcaccgagct 2520

gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga 2580

cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc 2640

cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat 2700

ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg 2760

cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga 2820

ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc 2880

cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg 2940

cacctggtgc atgacccgca agcccggtgc ctgaactccg tgtcaattat ttctcaaaca 3000

cgtacgcgta cgatgctcta gaatgctgat gggctagcaa aatcagcctc gactgtgcct 3060

tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt 3120

gccactccca ctgtcctttc ctaataaaat gaggaaattg catcacaaca ctcaacccta 3180

tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 3240

atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 3300

gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 3360

gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 3420

gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 3480

tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 3540

ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 3600

cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 3660

cgtgatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt 3720

cgacagcgtt tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt 3780

cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa 3840

agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga 3900

cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac 3960

gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat 4020

ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca 4080

aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt 4140

gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga 4200

tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt 4260

cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga 4320

ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt 4380

ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc 4440

gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt 4500

tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc 4560

cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga 4620

tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc 4680

aaaggaatag cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg 4740

cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct 4800

ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa 4860

tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 4920

caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc 4980

gtaatcattg tcattaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 5040

ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 5100

taccatctgg ccccagcgct gcgatgatac cgcgagaacc acgctcaccg gctccggatt 5160

tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 5220

ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 5280

atagtttgcg caacgttgtt gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg 5340

gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 5400

tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 5460

cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 5520

taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 5580

ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 5640

ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 5700

cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 5760

ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 5820

gaataagggc gacacggaaa tgttgaatac tcatattctt cctttttcaa tattattgaa 5880

gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 5940

aacaaatagg ggtcagtgtt acaaccaatt aaccaattct gaacattatc gcgagcccat 6000

ttatacctga atatggctca taacacccct tg 6032

<210>5

<211>1427

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

cgagatgatg tgctccaaac aggatccatg gtgagcaagg gcgaggagct gttcaccggg 60

gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 120

ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 180

ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 240

ttcagccgtt accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 300

ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 360

gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 420

aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 480

tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 540

atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 600

ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 660

cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 720

ctcggcatgg acgagctgta caagggaagc ggagagggca gaggaagtct gctaacatgc 780

ggtgacgtcg aggagaatcc tggacctatg accgagtaca agcccacggt gcgcctcgcc 840

acccgcgacg acgtcccccg ggccgtacgc accctcgccg ccgcgttcgc cgactacccc 900

gccacgcgcc acaccgtcga cccggaccgc cacatcgagc gggtcaccga gctgcaagaa 960

ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc 1020

gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc 1080

ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc 1140

ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg 1200

cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc 1260

gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac 1320

gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg 1380

tgcatgaccc gcaagcccgg tgcctgaacc gtgaaccagg ccagggt 1427

Claims (10)

1. A method of preparing a cell line comprising the steps of: performing gene editing on a B-16 melanoma cell by using a CRIS-PITCh system, inserting a coding gene of green fluorescent protein EGFP into the seventh exon end of IFNGR2 gene in the B-16 melanoma cell, and performing fusion expression on the coding gene of the green fluorescent protein EGFP and the IFNGR2 gene; the CRIS-PITCh system comprises a sgRNA expression cassette; the target sequence of the sgRNA is (a1) or (a2) as follows:

(a1) position 15881 and 15903 from the 5' end of SEQ ID NO. 1;

(a2) SEQ ID NO.1 at position 15936 and 15958 from the 5' end.

2. The method of claim 1, wherein: the CRIS-PITCh system comprises a carrier A and a carrier B; the vector A comprises a Cas9 expression cassette, an sgRNA expression cassette and a PITCh sgRNA expression cassette; the vector B comprises a donor fragment and homologous arms positioned at two ends of the donor fragment; the donor fragment is (b1) or (b2) as follows:

(b1) position 1595 and 2974 from the 5' end of SEQ ID NO. 4;

(b2) SEQ ID NO.5 positions 28 to 1407 from the 5' end.

3. The method of claim 2, wherein:

the homology arms at both ends of the donor fragment in (b1) are the upstream homology arm shown in position 1567-1586 from the 5 'end of SEQ ID NO.4 and the downstream homology arm shown in position 2975-2994 from the 5' end of SEQ ID NO. 4;

or, the homology arms at both ends of the donor fragment of (b2) are the upstream homology arm as shown in SEQ ID NO.5 from 1 to 20 at the 5 'end and the downstream homology arm as shown in SEQ ID NO.5 from 1408-1427 at the 5' end.

4. A cell line produced by the method of any one of claims 1 to 3.

5. The use of the cell line of claim 4 in the study of the function and expression regulation mechanism of the IFNGR2 gene.

6. Use of the cell line of claim 4 in the screening of drugs for targeting tumor immunity.

7. The sgRNA expression cassette or the CRIS-PITCh system of any one of claims 1 to 3.

8. Use of the sgRNA expression cassette of any one of claims 1 to 3 or the CRIS-PITCh system in the preparation of the cell line of claim 4.

9. The sgRNA expression cassette of any one of claims 1 to 3, or the CRIS-PITCh system, for use in drug screening for studying IFNGR2 gene function and expression regulation mechanism or targeted tumor immunity.

10. Use of the sgRNA expression cassette of any one of claims 1 to 3 or the CRIS-PITCh system in the preparation of a cell line for studying the function and expression regulation mechanism of the IFNGR2 gene or for drug screening for targeted tumor immunity.

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