CN108396027A - The sgRNA of CRISPR-Cas9 targeting knock out people colon-cancer cell DEAF1 genes and its specificity - Google Patents

Abstract

The invention discloses CRISPR/Cas9 targeting knock out people colon-cancer cell DEAF1 genes and its specificity sgRNA.It is first the sgRNA for obtaining selectively targeted DEAF1 genes Second Exon, base sequence is as shown in SEQ ID NO.1；Followed by the sgRNA of structure DEAF1 genes contains Cas9 albumen to slow virus carrier system, the system；Finally by 29 cells of CRISPR/Cas9 slow-virus infection people's intestinal cancer HT containing the sgRNA, the significantly reduced cell strain of DEAF1 protein expression levels is obtained.The present invention is simple with operating procedure, sgRNA targetings are good and high to DEAF1 genes cutting efficiency；In addition, constructed CRISPR/Cas9 slow virus systems, which have, knocks out efficient advantage, and energy specific knockdown DEAF1 genes, the people's colon-cancer cell for knocking out DEAF1 genes is obtained, to provide strong tool for the mechanism of action of DEAF1 in further research colon-cancer cell.

Description

CRISPR-Cas9 targeting knock out people colon-cancer cell DEAF1 genes and its specificity sgRNA

Technical field

The invention belongs to genetic engineering fields, more specifically to CRISPR/Cas9 targeting knock out people's colon-cancer cells The sgRNA of DEAF1 genes and its specificity.

Background technology

RBP-J is interacted and microtubule associated protein (DEAF1；C12orf52) it is a highly conserved albumen, albumen size For 36kD, there is no significant homology with any other protein.DEAF1 can interfere the transcription that Notch and RBP-J is mediated. DEAF1 quickly shuttles between nucleus and cytoplasm, it is most important that, it mediates the core output of RBP-J tubulin fibers. DEAF1, the RBP-J new as one interact albumen, by interfering transcription factor RBP-J to lower Notch.Notch gene codes A kind of highly conserved cell surface receptor, they adjust the development of a variety of biological cells such as from sea urchin to people.Notch signal shadows Ring multiple processes that cell normal morphology occurs, including the specialization of multipotency progenitor cells, Apoptosis, cell Proliferation and cell boundaries Formation.Transcription factor RBP-J has very important effect in regulating and controlling Notch signal paths, because it is inhibiting and is activating DNA target sequence is identified in compound.

Some researches show that the overexpression of DEAF1 increases the expression and downward of p53 and FBXW7 albumen in liver cancer cells The expression of cyclin D1, cyclin E, CDK2 and NF- κ B p65.These variations lead to the life of SMMC-7721 and HepG2 cells It is long to inhibit and induce G₀/G₁Phase cell-cycle arrest and apoptosis.It should be the result shows that it be by thin that DEAF1, which plays tumor inhibition effect, Born of the same parents period G₀/G₁Phase retardance is related with inducing cell apoptosis.DEAF1 genes participate in cell cycle regulation, since tumour is likely to be Because caused by cell hyperproliferation, therefore, it is necessary to study mechanism of action of the DEAF1 in different cells.

Intestinal cancer refers to betiding the carninomatosis class disease of enteron aisle, is malignant tumour common in gastrointestinal tract, and incidence is only second to Gastric cancer and the cancer of the esophagus.Currently, researcher has done numerous studies in terms of the mechanism of intestinal cancer, it was found that numerous participation intestinal cancer The approach and mechanism of progress, such as protooncogene activation, tumor suppressor gene inactivation (point mutation, rearrangement, missing) and chromosome abnormality, But current intestinal cancer occur with molecular mechanism involved in progress still there are many it is not yet clear, need further to study.

CRISPR/Cas systems are the important tools risen recent years for the specific DNA modification of target gene, are had fast The advantages such as fast, efficient and selectively targeted, have been applied to various modes biology, including people, mouse, rat, zebra fish, beautiful Hidden rhabditida, plant and bacterium.CRISPR RNA (crRNA) and transcriptional activation crRNA compounds to be formed of annealing can specifically be known Other genome sequence, guiding Cas9 endonucleases generate DNA double chain fracture in target fragment.This recognition complex can lead to Fusion crRNA and tracrRNA sequences formation sgRNA is crossed to be simplified.Have in the target sequence of genome be about the segment of 20bp with CrRNA or sgRNA complementary pairings；The trinucleotide region PAM (5 '-NGG-3 ') of target sequence end is spCas9 recognition sites, It is the key that realize shearing function.Can sgRNA accomplish that specificity, accurate targeting target gene are CRISPR/Cas9 specificity The prerequisite for knocking out target gene, misses the target and mistake targeting can all influence CRISPR/Cas9 and be struck to the specificity of target gene It removes, the sgRNA therefore, it is possible to design, prepare accuracy and selectively targeted target gene becomes CRISPR/Cas9 clpp genes The key technology removed.

The present invention utilizes CRISPR/Cas9 slow virus systems, obtains the people's colon-cancer cell for knocking out DEAF1 genes, for research The mechanism of action of DEAF1 provides powerful in colon-cancer cell.

Bibliography：

1)Horvath P,Barrangou R(January 2010)."CRISPR/Cas,the immune system of bacteria and archaea".Science327(5962):167–170.

2)Hale CR,Zhao P,Olson S,et al.(November 2009)."RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex".Cell 139(5):945–956.

3)Jinek,M.,Chylinski,K.,Fonfara,I.,Hauer,M.,Doudna,J.A.,and Charpentie E.(2012).A programmable dual-RNA-guided DNA endonuclease in adaptiv bacterial immunity.Science 337,816–821.

4)Hsu,P.D.,Scott,D.A.,Weinstein,J.A.,Ran,F.A.,Konermann,S.,Agarwala, V.,Li,Y.,Fine,E.J.,Wu,X.,Shalem,O.,et al.(2013).DNA targetingof RNA- guided Cas9 nucleases.Nat.Biotechnol.Published online July 21,2013.

5)Haihe Wang,Zhanchun Yang,Chunbo Liu,Shishun Huang,Hongzhi Wang, Yingli Chen,and Guofu Chen.(October 2014).RBP-J-interacting and tubulin- associated protein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma by activating the p53–Fbxw7pathway.Biochemical and Biophysical Research Communications 454(2014)71–77.

6)S.A.Wacker,C.Alvarado,G.von Wichert,et al.,DEAF1,a novel modulator of Notch signalling,acts via nuclear export of RBP-J,EMBO J.30(2011)43–56。

Invention content

The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of targeting knock out DEAF1 bases The sgRNA sequences of cause.

Another object of the present invention is to provide a kind of CRISPR/Cas9 slow virus of targeting knock out DEAF1 genes Systematic difference.

It is still another object of the present invention to provide the applications of people's colon-cancer cell HT-29 cells of targeting knock out DEAF1 genes.

The purpose of the invention is achieved by the following technical solution：A kind of sgRNA sequences of targeting knock out DEAF1 genes, choosing The DEAF1 sgRNA following from DNA sequence dna：

The sequence of DEAF1 sgRNA is as follows：

DEAF1 sgRNA oligo1：5’-CACCTGCCGCGTTCGCCACAGACG-3’；

DEAF1sgRNA oligo2：5’-AAACCGTCTGTGGCGAACGCGGCA-3’；

A kind of CRISPR/Cas9 slow virus systems of targeting knock out DEAF1 genes, contain above-mentioned targeting knock out DEAF1 bases The DNA sequence dna of the sgRNA of cause.

The structure of the CRISPR/Cas9 slow virus systems of the targeting knock out DEAF1 genes, includes the following steps：

(1) BsmBI digestion CRISPR/Cas9 slow virus carrier Lenti-CRISPRv2 are used, after obtaining digestion CRISPR/Cas9 slow virus carriers；

(2) by after the DNA sequence dna phosphorylation of the sgRNA of above-mentioned targeting knock out DEAF1 genes with the CRISPR/ after digestion Cas9 slow virus carriers connect, and obtain the CRISPR/Cas9 slow virus systems of targeting knock out DEAF1 genes.

DNA sequence dna described in step (2) is to move back oligonucleotide chain 1 (oligo1) and oligonucleotide chain 2 (oligo2) Fire obtains double-stranded sequence.The CRISPR/Cas9 slow virus system of the targeting knock out DEAF1 genes is preparing knockout DEAF1 Application in the cell strain of gene.A kind of cell strain knocking out DEAF1 genes, is by the targeting knock out DEAF1 genes CRISPR/Cas9 slow virus system transfections aim cell strains obtain.

The cell strain of the knockout DEAF1 genes, builds to obtain particular by following steps：

1) the CRISPR/Cas9 slow virus systems of the targeting knock out DEAF1 genes are wrapped by incasing cells Dress, obtains lentiviral particle；

2) lentiviral particle is infected into aim cell strain；

3) metainfective cell is spread into monoclonal, obtains the cell strain for knocking out DEAF1 genes.

The aim cell strain is preferably tumor cell line.

The tumor cell line is preferably colon-cancer cell strain.

The colon-cancer cell strain is preferably human colon carcinoma immortalized cells HT-29.

The cell strain of the knockout DEAF1 genes is DEAF1 gene delections or insertion nucleotide in HT-29 cells Obtained cell strain is any strain in following cell strain：

Cell strain 1 is that the gRNA CGTCTGTGGCGAACGCGGCA of wild type DEAF1 genes lack 4 bases, is sported CGTGGCGAACGCGGCA (sequencing result such as Fig. 6)；Cell strain 2 is the gRNA of wild type DEAF1 genes CGTCTGTGGCGAACGCGGCA lacks 5 bases, sports GTGGCGAACGCGGCA (sequencing result such as Fig. 7)；；Cell strain 3 It is gRNA CGTCTGTGGCGAACGCGGCA 6 bases of missing of wild type DEAF1 genes, sports TGGCGAACGCGGCA (sequencing result such as Fig. 8)；.The specific targeting knock out site of 3 kinds of DEAF1 genes and the comparison of wild type DEAF1 gene orders are such as Fig. 9.

The present invention has the following advantages and effects with respect to the prior art：

The present invention provides the sgRNA energy special target DEAF1 genes of DEAF1 genes, and it is slow to be built into CRISPR/Cas9 Virus system, the system energy special target knock out DEAF1 genes, the cell strain for knocking out DEAF1 genes are obtained, to be conducive to grind Study carefully the mechanism of action of the DEAF1 in cell strain.

Description of the drawings

Fig. 1 is the plasmid map of the vector plasmid lentiCRISPR v2 used in the embodiment of the present invention；

Fig. 2 is the plasmid figure of the expression vector plasmid lentiCRISPRv2-hDEAF1sg used in the embodiment of the present invention Spectrum；

Fig. 3 is the plasmid map of the packaging plasmid pLP-VSVG used in the embodiment of the present invention；

Fig. 4 is the plasmid map of the packaging plasmid psPAX2 used in the embodiment of the present invention；

Fig. 5 is that HT-29 cell virus infects the activity identification sequencer map containing DEAFlsgRNA；

Fig. 6 is the sequencer map of DEAF1 genes in the monoclonal HT-29 cell strains 1 for knock out DEAF1 genes；

Fig. 7 is the sequencer map of DEAF1 genes in the monoclonal HT-29 cell strains 2 for knock out DEAF1 genes；

Fig. 8 is the sequencer map of DEAF1 genes in the monoclonal HT-29 cell strains 3 for knock out DEAF1 genes；

Fig. 9 is the comparison diagram in the specific targeting knock out site and wild type DEAF1 gene orders of 3 kinds of DEAF1 genes.

Specific implementation mode

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.

Embodiment 1

1. knocking out DEAF1 plasmids using CRISPR/Cas9 technologies structure

1.1sgRNA oligonucleotide chains synthesize

Use CRISPR Photographing On-line tools (http：//crispr.mit.edu/) according to points-scoring system, DEAF1's The sgRNA of 1 20bp is designed on Second Exon, and is verified without non-specific gene by BLAST.The end of coding strand template 5 ' adds Add ACCG, the cohesive end formed after the addition of the end of noncoding strand template 3 ' AAAC, with BsmBI digestions is complementary, designs 1 couple of CRISPR Oligonucleotide chain is shown in Table 1.

1 DEAF1 target sites of table and sgRNA oligonucleotide sequences

1.2 vector construction

1.2.1 use 2 μ g Lenti-CRISPRv2 plasmids (being purchased from Addgene companies) of BsmBI digestions, 2h, 37 DEG C, enzyme The system of cutting is：

2μg(2μl)	Lenti-CRISPRv2
		1μl	BsmBI(NEB)
5μl	10X Cutsmart
		42μl	ddH2O
50μl	It is total

1.2.2 JaRa gene plastic recovery kit is used to purify digested plasmid product, by specification is operated.

1.2.3 phosphorylation and the sgRNA oligos that anneal, enzyme disjunctor system are：

1μl	Oligo1(100μM)
		1μl	Oligo2(100μM)
1μl	10X T4 link buffer solutions (NEB)
		6.5μl	ddH2O
0.5μl	T4 pNK(NEB)
		10μl	It is total

PCR instrument cycle of annealing：37 DEG C of 30min, 95 DEG C of maintenance 5min, per minute to reduce by 5 DEG C to 25 DEG C, 4 DEG C of maintenances

1.2.4 the Lenti-CRISPRv2 carriers after the Oligo double-strands of annealing formation and digestion are directly connected to, room temperature Under, 10min.

1.2.5 the plasmid after connection is converted into competent cell DH5 α, is uniformly applied in LB solid medium tablets, It is placed in 37 DEG C of incubators and cultivates 12-16 hours, single bacterium colony may occur in which.

The expansion of 1.3 picking single bacterium colonies, which is cultivated, and plasmid is small carries.

1.4 sequencing identification plasmid construction successes, and it is named as Lenti-CRISPRv2-hDEAFlsg.

1.5 knock out efficiency verification.

1) it is based on 5%CO2 with the DMEM in high glucose culture containing 10% fetal calf serum, 37 DEG C of constant temperature incubation 293T cells (are purchased from U.S.'s ATCC cell banks).Take logarithmic phase cell with 1 × 10⁵/ hole is inoculated into 24 orifice plate cultures.Wait for that cell fusion degree reaches 70% It is replaced with Opti-MEM culture mediums when~80%, corresponding CRISPR/Cas9 is knocked out into 0.8 μ g warps of plasmid after 1 hour Lipo2000 reagents are transfected into 293T cells, and as a child, fluorescence microscopy microscopic observation transfection, digestion is collected for transfection 48 Cell extracts cell genomic dna using genome DNA extracting reagent kit.

2) using each group cell genomic dna as template, sequence is targeted using primer (No.3~6 Seq) PCR amplification of table 2 Row.Carry out PCR with DEAF1-4-1 and DEAF1-4-2, obtain the segment a of 735bp, using a as template, with DEAF1-4-3 and DEAF1-4-4 carries out PCR, obtains the segment b of 404bp, and glue recycling is for being sequenced after the fragment electrophoretic, sequencing primer DEAF1- 4-3。

2 primer situation table of table

Primer	Primer sequence
		DEAF1-4-1	CAGGAATGGAGCTACAAGAGTG
DEAF1-4-2	GGCAGCTTCTACCTCAGCTG
		DEAF1-4-3	ACTGCACGTAAAAGGCTTTAC
DEAF1-4-4	CTCACAGAGCAGACGTTCAC

2. packing slow virus

100mm ware kinds enter 4X10⁶A 293TN cells, culture after (37 DEG C, 5%CO2), are replaced fresh culture for 24 hours, are turned Contaminate incasing cells：

A. 3 plasmids below mixing：1) 12 μ g of packaging plasmid psPAX2；10 μ g of helper plasmid pLP-VSVG；Express matter 22 μ g of grain Lenti-CRISPRv2-hDEAF1sg；CaCl is added₂250μl；DdH is added₂O is 500 μ l to total volume, and 37 DEG C put Set 20min；

B.2 500 μ l, 37 DEG C of placement 20min of X BES；

C. 500 μ l in a, tri- plasmid mixed liquors are added dropwise in b, and gently mixing；

D. careful that mixed liquor addition kind in c has in the 100mm wares of 293TN；Infection liquid is removed after cultivating 12h, is replaced 10ml fresh cultures produce virus liquid；After cultivating 48h, harvest in virus liquid to 50ml centrifuge tubes, and again into 100mm wares It adds 10ml culture mediums and produces virus again；After culture for 24 hours, virus liquid, room temperature centrifugation, 1000rpm, 10min are harvested again；Make With 0.22 μm of filter filter virus supernatant；Vial supernatant is fitted into and is surpassed from pipe, 4 DEG C, 100000g, 2h；It discards supernatant It is resuspended overnight with Opti-MEM after liquid；Reuse the virus that 0.22 μm of filter filtering is resuspended；After packing, -80 DEG C of preservations.

3. virus infection

1) the HT-29 cells (being purchased from U.S.'s ATCC cell banks) that will be infected are inoculated into six orifice plates, overnight, wait for that cell melts When right about 50%, culture medium is removed, replaces 2ml fresh cultures；

2) 20 μ l virus liquids are added per hole, 12 μ l polybrene polybrene are added, until final concentration of 6 μ g/ml；

3) after 37 DEG C of culture 12h, culture solution is removed, continues to cultivate 48h after replacing fresh medium；

4. screening stable cell line

After infection terminates 48h, puromycin (2.0 μ g/ml) is added to every hole, changes liquid every other day, and keep the fast of culture medium Purine mycin constant concentration, screening positive clone cell.Gained cell strain is named as HT-29-DEAF1.And use limiting dilution assay It selects HT-29-DEAF1 monoclonals and knocks out cell strain.

5. stable cell line is identified and gene targeting mode is identified

The monoclonal cell strain genomic DNA for knocking out DEAF1 using each group is separately designed as template outside for DEAF1 second The primer of son is shown, sequence such as Seq No.3~6 carry out two-wheeled nested PCR amplification, are surveyed to the second wheel PCR product of acquisition Sequence may be then to practice shooting successfully if bimodal situation occurs in sequencing result target practice location proximate.Select bimodal sample again PCR, TA is cloned into carrier T after the recycling of product glue, and picking positive colony is sequenced again after conversion, such as sequencing result The base insertion or base deletion of non-triple nearby occur for DEAF1 genes target site, lead to frame shift mutation, then can sentence Break as DEAF1 gene knockouts.It is active (Fig. 5) to identify sgRNA, and finds being lacked with DEAF1 genes for 2 types The stable cell line (Fig. 6-8) of mistake or insertion mutation.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications, Equivalent substitute mode is should be, is included within the scope of the present invention.

Sequence table

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tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 60

caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 120

ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 180

gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc 240

tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 300

ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 360

ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga 420

aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca 480

cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct 540

agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat 600

gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat 660

gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag 720

gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta 780

gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag 840

acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac 900

agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt 960

gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca 1020

aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg 1080

ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc 1140

agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg 1200

caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg 1260

gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa 1320

ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag 1380

atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta 1440

atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg 1500

gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat 1560

ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta 1620

ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc 1680

ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac 1740

agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca 1800

aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg 1860

ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 1920

tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat 1980

taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct 2040

gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg 2100

tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg 2160

gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg 2220

tggaaaggac gaaacaccgg agacggttgt aaatgagcac acaaaataca catgctaaaa 2280

tattatattc tatgaccttt ataaaatcaa ccaaaatctt ctttttaata actttagtat 2340

caataattag aatttttatg ttcctttttg caaactttta ataaaaatga gcaaaataaa 2400

aaaacgctag ttttagtaac tcgcgttgtt ttcttcacct ttaataatag ctactccacc 2460

acttgttcct aagcggtcag ctcctgcttc aatcattttt tgagcatctt caaatgttct 2520

aactccacca gctgctttaa ctaaagcatt gtctttaaca actgacttca ttagtttaac 2580

atcttcaaat gttgcacctg attttgaaaa tcctgttgat gttttaacaa attctaatcc 2640

agcttcaaca gctatttcac aagctttcat gatttcttct tttgttaata aacaattttc 2700

cataatacat ttaacaacat gtgatccagc tgcttttttt acagctttca tgtcttctaa 2760

aactaattca taatttttgt cttttaatgc accaatattt aataccatat caatttctgt 2820

tgcaccatct ttaattgctt cagaaacttc gaatgctttt gtagctgttg tgcatgcacc 2880

tagaggaaaa cctacaacat ttgttattcc tacatttgtg ccttttaata attctttaca 2940

atagcttgtt caatatgaat taacacaaac tgttgcaaaa tcaaattcaa ttgcttcatc 3000

acataattgt ttaatttcag ctttcgtagc atcttgtttt aataatgtgt gatctatata 3060

tttgtttagt ttcatttttt ctcctatata ttcattttta attttaattc tttaataatt 3120

tcgtctactt taactttagc gttttgaaca gattcaccaa cacctataaa ataaattttt 3180

agtttaggtt cagttccact tgggcgaaca gcaaatcatg acttatcttc taaataaaat 3240

tttagtaagt cttgtcctgg catattatac attccatcga tgtagtcttc aacattaaca 3300

actttaagtc cagcaatttg agttaagggt gttgctctca atgatttcat taatggttca 3360

atttttaatt tcttttcttc tggtttaaaa ttcaagttta aagtgaaagt gtaatatgca 3420

cccatttctt taaataaatc ttctaaatag tctactaatg ttttattttg ttttttataa 3480

aatcaagcag cctctgctat taatatagaa gcttgtattc catctttatc tctagctgag 3540

tcatcaatta catatccata actttcttca taagcaaaaa caaaatttaa tccgttatct 3600

tcttctttag caatttctct acccattcat ttaaatccag ttaaagtttt tacaatatta 3660

actccatatt tttcatgagc gattctatca cccaaatcac ttgttacaaa acttgaatat 3720

agagccggat tttttggaat gctatttaag cgttttagat ttgataattt tcaatcaatt 3780

aaaattggtc ctgtttgatt tccatctaat cttacaaaat gaccatcatg ttttattgcc 3840

attccaaatc tgtcagcatc tgggtcattc ataataataa tatctgcatc atgtttaata 3900

ccatattcaa gcggtatttt tcatgcagga tcaaattctg gatttggatt tacaacattt 3960

ttaaatgttt catcttcaaa tgcatgctct tcaacctcaa taacgttata tcctgattca 4020

cgtaatattt ttggggtaaa tttagttcct gttccattaa ctgcgctaaa aataattttt 4080

aaatcttttt tagcttcttg ctcttttttg tacgtctctg ttttagagct agaaatagca 4140

agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 4200

tgaattcgct agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc 4260

agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg 4320

tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct 4380

ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt 4440

tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa 4500

gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga 4560

cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat 4620

caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg 4680

gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca 4740

agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga 4800

gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt 4860

ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt 4920

ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa 4980

gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa 5040

gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc 5100

cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga 5160

aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc 5220

cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa 5280

actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg 5340

cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag 5400

cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa 5460

gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct 5520

gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat 5580

tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat 5640

ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg 5700

gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct 5760

gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat 5820

cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc 5880

ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga 5940

caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc 6000

caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga 6060

gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga 6120

gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca 6180

gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt 6240

ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga 6300

caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct 6360

gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt 6420

cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag 6480

ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct 6540

gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac 6600

ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca 6660

cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt 6720

ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat 6780

ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg 6840

gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa 6900

cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta 6960

cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc 7020

tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa 7080

ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg 7140

gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc 7200

cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga 7260

aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta 7320

cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt 7380

gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca 7440

cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa 7500

gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc 7560

caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat 7620

gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat 7680

cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt 7740

gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg 7800

cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa 7860

gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt 7920

gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct 7980

gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga 8040

agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct 8100

gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg 8160

aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga 8220

gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa 8280

gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc 8340

cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag 8400

agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc 8460

cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct 8520

ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc 8580

tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa 8640

gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa 8700

acaagccgga gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct 8760

cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta 8820

ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca 8880

agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg 8940

cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga 9000

gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga 9060

aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt 9120

ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc 9180

ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt 9240

ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac 9300

ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga 9360

ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 9420

tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 9480

ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 9540

gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 9600

caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 9660

actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 9720

ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac 9780

ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 9840

tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 9900

gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac 9960

aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt 10020

cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag 10080

atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc 10140

ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga 10200

tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg 10260

atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 10320

ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 10380

atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 10440

gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc 10500

tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc 10560

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 10620

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 10680

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 10740

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 10800

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 10860

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 10920

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg 10980

aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 11040

agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 11100

agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 11160

cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 11220

ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga 11280

ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt 11340

ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat 11400

aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca 11460

ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg 11520

acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg 11580

cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg 11640

acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc 11700

cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg 11760

ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg 11820

attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct 11880

ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta 11940

ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 12000

ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 12060

gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt 12120

gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 12180

cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 12240

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 12300

gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 12360

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 12420

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 12480

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 12540

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 12600

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 12660

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 12720

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 12780

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 12840

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 12900

cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 12960

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 13020

aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 13080

aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 13140

actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 13200

taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 13260

gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 13320

tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 13380

ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 13440

accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 13500

agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 13560

acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 13620

tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 13680

cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 13740

tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 13800

ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 13860

gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 13920

tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 13980

ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 14040

gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 14100

cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 14160

gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 14220

ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga 14280

tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct 14340

gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac 14400

aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct 14460

gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata 14520

gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact 14580

tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat 14640

gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta 14700

tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc 14760

tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg 14820

ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgg 14873

<210> 8

<211> 13012

<212> DNA

<213>Artificial sequence (ARTIFICAL SYNTHESIS)

<400> 8

tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 60

caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 120

ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 180

gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc 240

tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 300

ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 360

ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga 420

aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca 480

cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct 540

agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat 600

gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat 660

gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag 720

gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta 780

gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag 840

acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac 900

agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt 960

gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca 1020

aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg 1080

ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc 1140

agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg 1200

caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg 1260

gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa 1320

ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag 1380

atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta 1440

atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg 1500

gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat 1560

ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta 1620

ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc 1680

ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac 1740

agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca 1800

aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg 1860

ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 1920

tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat 1980

taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct 2040

gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg 2100

tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg 2160

gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg 2220

tggaaaggac gaaacacctg ccgcgttcgc cacagacggt tttagagcta gaaatagcaa 2280

gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 2340

gaattcgcta gctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca 2400

gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt 2460

gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt 2520

tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt 2580

cgcaacgggt ttgccgccag aacacaggac cggttctaga gcgctgccac catggacaag 2640

aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac 2700

gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc 2760

aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg 2820

ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa 2880

gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag 2940

tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg 3000

gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg 3060

gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag 3120

ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag 3180

ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc 3240

agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa 3300

aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc 3360

ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa 3420

ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc 3480

gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc 3540

gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag 3600

agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg 3660

cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt 3720

gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg 3780

gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg 3840

accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg 3900

cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc 3960

ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc 4020

tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac 4080

aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc 4140

aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag 4200

ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag 4260

cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag 4320

ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg 4380

gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac 4440

aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg 4500

acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc 4560

gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc 4620

cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg 4680

aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc 4740

tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac 4800

attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg 4860

gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg 4920

gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg 4980

atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac 5040

acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac 5100

gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct 5160

cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac 5220

cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg 5280

cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc 5340

gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa 5400

acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac 5460

gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg 5520

tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac 5580

gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag 5640

ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc 5700

aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg 5760

aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc 5820

gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 5880

cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 5940

ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag 6000

aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg 6060

ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 6120

ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 6180

gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 6240

ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga 6300

aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 6360

aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 6420

cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 6480

gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 6540

gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc 6600

ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg 6660

gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 6720

cagctgggag gcgacaagcg acctgccgcc acaaagaagg ctggacaggc taagaagaag 6780

aaagattaca aagacgatga cgataaggga tccggcgcaa caaacttctc tctgctgaaa 6840

caagccggag atgtcgaaga gaatcctgga ccgaccgagt acaagcccac ggtgcgcctc 6900

gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac 6960

cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa 7020

gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc 7080

gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag 7140

atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa 7200

ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc 7260

tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg 7320

gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc 7380

tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc 7440

tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat 7500

tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 7560

ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 7620

tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 7680

caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 7740

accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 7800

ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 7860

tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc 7920

tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 7980

ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 8040

acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca 8100

aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc 8160

actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga 8220

tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct 8280

tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat 8340

ccctcagacc cttttagtca gtgtggaaaa tctctagcag ggcccgttta aacccgctga 8400

tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 8460

tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 8520

tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 8580

ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct 8640

gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca 8700

ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 8760

gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 8820

caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 8880

cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 8940

tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 9000

acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 9060

gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga 9120

atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 9180

gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca 9240

gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc 9300

ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 9360

tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag 9420

gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt 9480

tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata 9540

atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac 9600

cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga 9660

cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc 9720

ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga 9780

cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc 9840

ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc 9900

cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga 9960

ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg 10020

gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat 10080

tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 10140

tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 10200

tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 10260

aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 10320

ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 10380

ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 10440

cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 10500

tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 10560

aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 10620

aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 10680

tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 10740

ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 10800

cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 10860

ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 10920

ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 10980

gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 11040

agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 11100

cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 11160

aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 11220

aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 11280

ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 11340

aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 11400

ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 11460

agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 11520

cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 11580

ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 11640

gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 11700

cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 11760

cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 11820

ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 11880

catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 11940

tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 12000

ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 12060

catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 12120

cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 12180

cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 12240

acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 12300

ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 12360

tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat 12420

cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg 12480

ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca 12540

aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg 12600

cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag 12660

taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 12720

acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 12780

acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 12840

ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 12900

attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 12960

gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat gg 13012

<210> 9

<211> 6507

<212> DNA

<213>Artificial sequence (ARTIFICAL SYNTHESIS)

<400> 9

gagcttggcc cattgcatac gttgtatcca tatcataata tgtacattta tattggctca 60

tgtccaacat taccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt 120

acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 180

ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 240

cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 300

actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc 360

aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 420

acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag 480

tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 540

gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 600

aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc 660

agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc 720

catagaagac accgggaccg atccagcctc cggtcgaccg atcctgagaa cttcagggtg 780

agtttgggga cccttgattg ttctttcttt ttcgctattg taaaattcat gttatatgga 840

gggggcaaag ttttcagggt gttgtttaga atgggaagat gtcccttgta tcaccatgga 900

ccctcatgat aattttgttt ctttcacttt ctactctgtt gacaaccatt gtctcctctt 960

attttctttt cattttctgt aactttttcg ttaaacttta gcttgcattt gtaacgaatt 1020

tttaaattca cttttgttta tttgtcagat tgtaagtact ttctctaatc actttttttt 1080

caaggcaatc agggtatatt atattgtact tcagcacagt tttagagaac aattgttata 1140

attaaatgat aaggtagaat atttctgcat ataaattctg gctggcgtgg aaatattctt 1200

attggtagaa acaactacac cctggtcatc atcctgcctt tctctttatg gttacaatga 1260

tatacactgt ttgagatgag gataaaatac tctgagtcca aaccgggccc ctctgctaac 1320

catgttcatg ccttcttctc tttcctacag ctcctgggca acgtgctggt tgttgtgctg 1380

tctcatcatt ttggcaaaga attcctcgac ggatccctcg aggaattctg acactatgaa 1440

gtgccttttg tacttagcct ttttattcat tggggtgaat tgcaagttca ccatagtttt 1500

tccacacaac caaaaaggaa actggaaaaa tgttccttct aattaccatt attgcccgtc 1560

aagctcagat ttaaattggc ataatgactt aataggcaca gccttacaag tcaaaatgcc 1620

caagagtcac aaggctattc aagcagacgg ttggatgtgt catgcttcca aatgggtcac 1680

tacttgtgat ttccgctggt atggaccgaa gtatataaca cattccatcc gatccttcac 1740

tccatctgta gaacaatgca aggaaagcat tgaacaaacg aaacaaggaa cttggctgaa 1800

tccaggcttc cctcctcaaa gttgtggata tgcaactgtg acggatgccg aagcagtgat 1860

tgtccaggtg actcctcacc atgtgctggt tgatgaatac acaggagaat gggttgattc 1920

acagttcatc aacggaaaat gcagcaatta catatgcccc actgtccata actctacaac 1980

ctggcattct gactataagg tcaaagggct atgtgattct aacctcattt ccatggacat 2040

caccttcttc tcagaggacg gagagctatc atccctggga aaggagggca cagggttcag 2100

aagtaactac tttgcttatg aaactggagg caaggcctgc aaaatgcaat actgcaagca 2160

ttggggagtc agactcccat caggtgtctg gttcgagatg gctgataagg atctctttgc 2220

tgcagccaga ttccctgaat gcccagaagg gtcaagtatc tctgctccat ctcagacctc 2280

agtggatgta agtctaattc aggacgttga gaggatcttg gattattccc tctgccaaga 2340

aacctggagc aaaatcagag cgggtcttcc aatctctcca gtggatctca gctatcttgc 2400

tcctaaaaac ccaggaaccg gtcctgcttt caccataatc aatggtaccc taaaatactt 2460

tgagaccaga tacatcagag tcgatattgc tgctccaatc ctctcaagaa tggtcggaat 2520

gatcagtgga actaccacag aaagggaact gtgggatgac tgggcaccat atgaagacgt 2580

ggaaattgga cccaatggag ttctgaggac cagttcagga tataagtttc ctttatacat 2640

gattggacat ggtatgttgg actccgatct tcatcttagc tcaaaggctc aggtgttcga 2700

acatcctcac attcaagacg ctgcttcgca acttcctgat gatgagagtt tattttttgg 2760

tgatactggg ctatccaaaa atccaatcga gcttgtagaa ggttggttca gtagttggaa 2820

aagctctatt gcctcttttt tctttatcat agggttaatc attggactat tcttggttct 2880

ccgagttggt atccatcttt gcattaaatt aaagcacacc aagaaaagac agatttatac 2940

agacatagag atgaaccgac ttggaaagta actcaaatcc tgcacaacag attcttcatg 3000

tttggaccaa atcaacttgt gataccatgc tcaaagaggc ctcaattata tttgagtttt 3060

taatttttat gaaaaaaaaa aaaaaaaacg gaattcctcg agggatccgt cgaggaattc 3120

actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct 3180

cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc 3240

cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg 3300

aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag 3360

aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa 3420

ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc 3480

catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt 3540

tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc 3600

tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga cggatcggcc 3660

gcaattcgta atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 3720

acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 3780

actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 3840

gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 3900

cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 3960

tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 4020

gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 4080

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 4140

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 4200

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 4260

ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 4320

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 4380

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 4440

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 4500

ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 4560

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 4620

ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 4680

cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 4740

gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 4800

aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 4860

acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 4920

gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 4980

cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 5040

cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 5100

tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 5160

cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 5220

gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 5280

cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 5340

ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 5400

gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 5460

taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 5520

gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 5580

acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 5640

aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 5700

cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 5760

atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 5820

gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc 5880

agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag 5940

accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg 6000

gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca 6060

tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa 6120

gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg 6180

aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta 6240

accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg 6300

ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg 6360

aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga 6420

cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattggag 6480

ctccaccgcg gtggcggccg ctctaga 6507

<210> 10

<211> 10668

<212> DNA

<213>Artificial sequence (ARTIFICAL SYNTHESIS)

<400> 10

gcggctcgcc gcgcctcgac agccgccggc gctccggggg ccgccgcgcc cctcccccga 60

gccctccccg gcccgaggcg gccccgcccc gcccggcacc cccacctgcc gccacccccc 120

gcccggcacg gcgagccccg cgccacgccc cgtacggagc cccgcacccg aagccgggcc 180

gtgctcagca actcggggag gggggtgcag ggggggttgc agcccgaccg acgcgcccac 240

accccctgct caccccccca cgcacacacc ccgcacgcag cctttgttcc cctcgcagcc 300

ccccccgcac cgcggggcac cgcccccggc cgcgctcccc tcgcgcacac tgcggagcgc 360

acaaagcccc gcgccgcgcc cgcagcgctc acagccgccg ggcagcgcgg agccgcacgc 420

ggcgctcccc acgcacacac acacgcacgc accccccgag ccgctccccc cgcacaaagg 480

gccctcccgg agcccctcaa ggctttcacg cagccacaga aaagaaacaa gccgtcatta 540

aaccaagcgc taattacagc ccggaggaga agggccgtcc cgcccgctca cctgtgggag 600

taacgcggtc agtcagagcc ggggcgggcg gcgcgaggcg gcggcggagc ggggcacggg 660

gcgaaggcag cgcgcagcga ctcccgcccg ccgcgcgctt cgctttttat agggccgccg 720

ccgccgccgc ctcgccataa aaggaaactt tcggagcgcg ccgctctgat tggctgccgc 780

cgcacctctc cgcctcgccc cccccccccc cccgccccca tcgctgcaca aaataattaa 840

aaaataaata aatacaaaat tgggggtggg gagggggggg agatggggag agtgaagcag 900

aacgtggggc tcacctcgac catggtaata gcgatgacta atacgtagat gtactgccaa 960

gtaggaaagt cccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca 1020

ttgacgtcaa tagggggcgt acttggcata tgatacactt gatgtactgc caagtgggca 1080

gtttaccgta aatactccac ccattgacgt caatggaaag tccctattgg cgttactatg 1140

ggaacatacg tcattattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg 1200

ccatttaccg taagttatgt aacgcggaac tccatatatg ggctatgaac taatgacccc 1260

gtaattgatt actattaata actagtcaat aatcaatgtc gacccaggtg gcacttttcg 1320

gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 1380

gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 1440

tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 1500

tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 1560

gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 1620

acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 1680

tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 1740

gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 1800

tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 1860

accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 1920

ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 1980

agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 2040

gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 2100

ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 2160

tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 2220

ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 2280

gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 2340

acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 2400

aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 2460

atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 2520

gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 2580

tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca 2640

ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 2700

ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 2760

ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 2820

aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 2880

cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 2940

gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 3000

ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 3060

cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 3120

tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 3180

cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 3240

cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gcggatccag 3300

acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat 3360

gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata 3420

aacaagttag ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga 3480

atagctcaga ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat 3540

ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg 3600

ggactatggt tgctgactaa ttgagatgcg gatccgctgg cacgacaggt ttcccgactg 3660

gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca 3720

ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt 3780

tcacacagga aacagctatg accatgatta cgccaagctt gggctgcagg tcgagggatc 3840

tccataagag aagagggaca gctatgactg ggagtagtca ggagaggagg aaaaatctgg 3900

ctagtaaaac atgtaaggaa aattttaggg atgttaaaga aaaaaataac acaaaacaaa 3960

atataaaaaa aatctaacct caagtcaagg cttttctatg gaataaggaa tggacagcag 4020

ggggctgttt catatactga tgacctcttt atagccaacc tttgttcatg gcagccagca 4080

tatgggcata tgttgccaaa ctctaaacca aatactcatt ctgatgtttt aaatgatttg 4140

ccctcccata tgtccttccg agtgagagac acaaaaaatt ccaacacact attgcaatga 4200

aaataaattt cctttattag ccagaagtca gatgctcaag gggcttcatg atgtccccat 4260

aatttttggc agagggaaaa agatctgcta gctatagttc tagaggtacc ggttgtttcg 4320

agcttatagc aaaatccttt ccaagccctg tcttattctt ctaggtatgt ggcgaatagc 4380

tctacaagct ccttgtacta cttctataac cctatctgtc ccctcagcta ctgctatggc 4440

tgtggcattg agcaagctaa cagcactatt ctttagctcc tgactccaat attgtaggag 4500

attccaccaa tatttgaggg cttcccaccc cctgcgtccc agaagttcca caatcctcgt 4560

tacaatcaag agtaagtctc tcaagcggtg gtagctgaag aggcacaggc tccgcagatc 4620

gtcccagata agtgccaagg atccgttcac taatcgaatg gatctgtctc tgtctctctc 4680

tccaccttct tcttctattc cttcgggcct gtcgggtccc ctcggggttg ggaggtgggt 4740

ctgaaacgat aatggtgaat atccctgcct aactctattc actatagaaa gtacagcaaa 4800

aactattctt aaacctacca agcctcctac tatcattatg aataatttta tataccacag 4860

ccaatttgtt atgttaaacc aattccacaa acttgcccat ttatctaatt ccaataattc 4920

ttgttcattc ttttcttgct ggttttgcga ttcttcaatt aaggagtgta ttaagcttgt 4980

gtaattgtta atttctctgt cccactccat ccaggtcgtg tgattccaaa tctgttccag 5040

agatttatta ctccaactag cattccaagg cacagcagtg gtgcaaatga gttttccaga 5100

gcaaccccaa atccccagga gctgttgatc ctttaggtat ctttccacag ccaggattct 5160

tgcctggagc tgcttgatgc cccagactgt gagttgcaac agatgctgtt gcgcctcaat 5220

agccctcagc aaattgttct gctgctgcac tataccagac aataattgtc tggcctgtac 5280

cgtcagcgtc attgacgctg cgcccatagt gcttcctgct gctcccaaga acccaaggaa 5340

caaagctcct attcccactg ctcttttttc tctctgcacc actcttctct ttgccttggt 5400

gggtgctact cctaatggtt caatttttac tactttatat ttatataatt cacttctcca 5460

attgtccctc atatctcctc ctccaggtct gaagatcagc ggccattttc ctatattcta 5520

tgattactat ggaccacaca actattgcta ttattattgc tactactaat gctactattg 5580

ctactattat aggttgcatt acatgtacta cttactgctt tgatagagaa gcttgatgag 5640

tctgactgtt ctgatgagct cttcgtcgct gtctccgctt cttcctgcca taggagatgc 5700

ctaaggcttt tgtcatgaaa caaacttggc aatgaaagca acacttttta caatagcaat 5760

tggtacaagc agttttaggc tgacttcctg gatgcttcca gggctctagt ctaggatcta 5820

ctggctccat ttcttgctct cctctgtcga gtaacgccta ttctgctatg tcgacaccca 5880

attctgaaat ggataaacag cagttgttgc agaattccat gtgttaatcc tcatcctgtc 5940

tacttgccac acaatcatca cctgccatct gttttccata atccctgatg atctttgctt 6000

ttcttcttgg cactactttt atgtcactat tatcttgtat tactactgcc ccttcacctt 6060

tccagaggag ctttgctggt cctttccaaa ctggatctct gctgtccctg taataaaccc 6120

gaaaattttg aatttttgta atttgttttt gtaattcttt agtttgtatg tctgttgcta 6180

ttatgtctac tattctttcc cctgcactgt accccccaat cccccctttt cttttaaaat 6240

tgtggatgaa tactgccatt tgtactgctg tcttaagatg ttcagcctga tctcttacct 6300

gtcctataat tttctttaat tctttattca tagattctat tactccttga ctttggggat 6360

tgtagggaat gccaaattcc tgcttgatcc ccgcccacca acaggcggcc ttaactgtag 6420

tactggtgaa attgctgcca ttgtctgtat gtactgtttt tactggccat cttcctgcta 6480

attttaagag gaagtatgct gtttcttgcc ctgtctctgc tggaattact tctgcttcta 6540

tatatccact ggctacatga actgctacca agataacttt tccttctaaa tgtgtacaat 6600

ctagctgcca tattcctggg ctacagtcta cttgtccatg catggcttcc ccttttagct 6660

gacatttatc acagctggct actatttctt ttgctactac aggtggtagg ttaaaatcac 6720

tagccattgc tctccaatta ctgtgatatt tctcatgttc ttcttgggcc ttatctattc 6780

catctaaaaa tagtactttc ctgattccag cactgaccaa tttatctact tgttcatttc 6840

ctccaattcc tttgtgtgct ggtacccatg ccaggtagac tttttccttt tttattaact 6900

gctctattat ttgactgact aactctgatt cactcttatc tggttgtgct tgaatgattc 6960

ccaatgcata ttgtgagtct gtcactatgt ttacttctaa tcccgaatcc tgcaaagcta 7020

gatgaattgc ttgtaactca gtcttctgat ttgttgtgtc cgttaggggg acaacttttt 7080

gtcttcctct gtcagttaca tatcctgctt ttcctaattt agtttcccta ttggctgccc 7140

catctacata gaaagtttct gctcctatta tgggttcttt ctctaactgg taccataact 7200

tcactaaggg aggggtattg acaaactccc actcaggaat ccaggtggct tgccaatact 7260

ctgtccacca tgcttcccat gtttcctttt gtatgggtaa tttaaattta ggagtctttc 7320

cccatattac tatgctttct gtggctattt tttgtactgc ctctgttaat tgtttcacat 7380

cattagtgtg ggcacccttc attcttgcat actttcctgt tttcagattt ttaaatggct 7440

cttgataaat ttgatatgtc cattggcctt gcccctgctt ctgtatttct gctattaagt 7500

cttttgatgg gtcataatac actccatgta ccggttcttt tagaatctcc ctgttttctg 7560

ccagttctag ctctgcttct tctgttagtg gtactacttc tgttagtgct ttggttcccc 7620

taagaagttt acataattgc cttactttaa tccctgcata aatctgactt gcccaattca 7680

attttcccac taatttctgt atgtcattga cagtccagct gtccttttct ggcagcacta 7740

taggctgtac tgtccattta tcaggatgga gttcataacc catccaaagg aatggaggtt 7800

ctttctgatg ttttttgtct ggtgtggtaa atccccacct caacagatgt tgtctcagtt 7860

cctctatttt tgttctatgc tgccctattt ctaagtcaga tcctacatac aaatcatcca 7920

tgtattgata gatgactatg tctggatttt gttttctaaa aggctctaag atttttgtca 7980

tgctacactg gaatattgct ggtgatcctt tccatccctg tggaagcaca ttgtactgat 8040

atctaatccc tggtgtctca ttgtttatac taggtatggt aaatgcagta tacttcctga 8100

agtctttatc taagggaact gaaaaatatg catcgcccac atccagtact gttactgatt 8160

ttttctgttt taaccctgca ggatgtggta ttcctaattg aacttcccag aaatcttgag 8220

ttctcttatt aagttctctg aaatctacta attttctcca tttagtactg tcttttttct 8280

ttatggcaaa tactggagta ttgtatggat tttcaggccc aatttttgaa atttttcctt 8340

ccttttccat ttctgtacaa atttctacta atgcttttat tttttcttct gtcaatggcc 8400

attgtttaac ttttgggcca tccattcctg gctttaattt tactggtaca gtctcaatag 8460

gactaatggg aaaatttaaa gtgcagccaa tctgagtcaa cagatttctt ccaattatgt 8520

tgacaggtgt aggtcctact aatactgtac ctatagcttt atgtccgcag atttctatga 8580

gtatctgatc atactgtctt actttgataa aacctccaat tccccctatc atttttggtt 8640

tccatcttcc tggcaaattc atttcttcta atactgtatc atctgctcct gtatctaata 8700

gagcttcctt taattgcccc cctatcttta ttgtgacgag gggtcgctgc caaagagtga 8760

tctgagggaa gctaaaggat acagttcctt gtctatcggc tcctgcttct gagagggagt 8820

tgttgtctct tccccaaacc tgaagctctc ttctggtggg gctgttggct ctggtctgct 8880

ctgaagaaaa ttccctggcc ttcccttgtg ggaaggccag atcttcccta aaaaattagc 8940

ctgtctctca gtacaatctt tcatttggtg tccttccttt ccacatttcc aacagccctt 9000

tttcctaggg gccctgcaat ttttggctat gtgcccttct ttgccacaat tgaaacactt 9060

aacagtcttt ctttggttcc taaaattgcc tttctgtatc attatggtag ctggatttgt 9120

tacttggctc attgcttcag ccaaaactct tgctttatgg ccgggtcccc ccactccctg 9180

acatgctgtc atcatttctt ctagtgtcgc tcctggtccc aatgctttta aaatagtctt 9240

acaatctggg ttcgcatttt ggaccaacaa ggtttctgtc atccaatttt ttacctcttg 9300

tgaagcttgc tcggctctta gagttttata gaatcggtct acatagtctc taaagggttc 9360

ctttggtcct tgtcttatgt ccagaatgct ggtagggcta tacattctta ctattttatt 9420

taatcccagg attatccatc ttttatagat ttctcctact gggataggtg gattatgtgt 9480

catccatcct atttgttcct gaagggtact agtagttcct gctatgtcac ttccccttgg 9540

ttctctcatc tggcctggtg caataggccc tgcatgcact ggatgcactc tatcccattc 9600

tgcagcttcc tcattgatgg tctcttttaa catttgcatg gctgcttgat gtccccccac 9660

tgtgtttagc atggtgttta aatcttgtgg ggtggctcct tctgataatg ctgaaaacat 9720

gggtatcact tctgggctga aagccttctc ttctactact tttacccatg catttaaagt 9780

tctaggtgat atggcctgat gtaccatttg cccctggatg ttctgcacta tagggtaatt 9840

ttggctgacc tgattgctgt gtcctgtgtc agctgctgct tgctgtgctt ttttcttact 9900

tttgttttgc tcttcctcta tcttgtctaa agcttccttg gtgtctttta tctctatcct 9960

ttgatgcaca caatagaggg ttgctactgt attatataat gatctaagtt cttctgatcc 10020

tgtctgaagg gatggttgta gctgtcccag tatttgtcta cagccttctg atgtttctaa 10080

caggccagga ttaactgcga atcgttctag ctccctgctt gcccatacta tatgttttaa 10140

tttatatttt ttctttcccc ctggccttaa ccgaattttt tcccatcgat ctaattctcc 10200

cccgcttaat actgacgctc tcgcacccat ctctcaccag tcgccgcccc tcgcctcttg 10260

ccgtgcgcgt caacgcggcc cgaattcttt gccaaaatga tgagacagca caataaccag 10320

cacgttgccc aggagctgta ggaaaaagaa gaaggcatga acatggttag cagaggctct 10380

agagccgccg gtcacacgcc agaagccgaa ccccgccctg ccccgtcccc cccgaaggca 10440

gccgtccccc cgcggacagc cccgaggctg gagagggaga aggggacggc ggcgcggcga 10500

cgcacgaagg ccctccccgc ccatttcctt cctgccggcg ccgcaccgct tcgccccgcg 10560

cccgctagag ggggtgcggc ggcgcctccc agatttcggc tccgcacaga tttgggacaa 10620

aggaagtccc tgcgccctct cgcacgatta ccataaaagg caatggct 10668

Claims (7)

1. the sgRNA of a kind of CRISPR/Cas9 targeting knock outs people colon-cancer cell DEAF1 genes and its specificity, which is characterized in that Design obtains the sgRNA of selectively targeted DEAF1 genes Second Exon first；Secondly the sgRNA of structure DEAF1 genes is to slowly Virus carrier system；Then this slow virus carrier system is infected into people's intestinal cancer HT-29 cells, obtained DEAF1 gene knockouts are thin Born of the same parents' strain.

2. the sgRNA of selectively targeted DEAF1 genes Second Exon according to claim 1, DNA sequence dna such as SEQ Shown in ID NO.1, target sequences of the sgRNA on DEAF1 genes is unique.

3. the sgRNA of selectively targeted DEAF1 genes Second Exon according to claim 2, primer include

It is located at the primer pair on Second Exon for the target spot of DEAF1 genes：

DEAF1sgRNAoligo1：As shown in SEQ ID NO.2；

DEAF1sgRNAoligo2：As shown in SEQ ID NO.3.

It is a kind of targeting knock out 4. the sgRNA of structure DEAF1 genes according to claim 1 is to slow virus carrier system The CRISPR/Cas9 recombinant expression slow virus carriers Lenti-CRISPRv2-hDEAF1sg of DEAF1 genes, it is characterised in that：Contain There is the recombinant expression carrier of the sgRNA and Cas9 albumen of selectively targeted DEAF1 genes Second Exon.

5. SEQ ID NO in the sequence of the skeleton carrier of recombinant expression carrier according to claim 4 such as sequence table：8 institutes Show.

6. CRISPR/Cas9 targeting knock outs people colon-cancer cell DEAF1 genes according to claim 1 and its specificity SgRNA, it is characterised in that include the following steps：

(1) sgRNA sequences are provided, target sequences of the sgRNA on DEAF1 genes meets the series arrangement rule of 5 '-N (19) G Then, target sequences of the sgRNA on DEAF1 genes is located at the exon of gene, targets of the sgRNA on DEAF1 genes Sequence is located on the common exon of different various shear patterns, and target sequences of the sgRNA on DEAF1 genes is unique , and target site sequences of the sgRNA on DEAF1 be as shown in sequence table SEQ ID NO.1 sequences, in the sgRNA 5 '-ends of the target site sequence on DEAF1 obtain positive oligonucleotides i.e. Forward oligo plus CACCG sequent synthesis； The complementary strand of target site sequences of the sgRNA on DEAF1 is obtained, and at 5 '-ends of complementary strand plus AAAC sequences, 3 '-ends In addition C, synthesis obtains reverse oligonucleotide i.e. Reverse oligo；By the complementary sgRNA oligonucleotides of 1 couple of synthesis Forward oligo and Reverse oligo are denaturalized, anneal in pairs, and U6 carrier for expression of eukaryon can be connected by being formed after annealing Double-strand sgRNA oligonucleotides；

(2) sequence Lenti-CRISPRv2 plasmids as shown in sequence table SEQ ID NO.8 are linearized；By the double-strand of annealing SgRNA oligonucleotides connect acquisition and carry with the expression vector Lenti-CRISPRv2 of the carrying Cas9 genes of linearisation contains phase The sgRNA oligonucleotides of target sequence and the expression vector Lenti-CRISPRv2-hDEAF1sg plasmids of Cas9 genes are answered, is converted Competent bacteria simultaneously applies Amp+ tablets, and picking monoclonal simultaneously identifies positive colony with universal primer U6 by sequencing, and to institute It states positive colony and shakes bacterium, extraction plasmid；

(3) expression vector that the sequence of sgRNA oligonucleotides and Cas9 genes is SEQ ID NO.9 is carried described in Lenti-CRISPRv2-hDEAF1sg plasmids, the pLP-VSVG that sequence is SEQ ID NO.10 and SEQ ID NO.11 (AddGene 8454) and psPAX2 (AddGene 12260) packaging plasmids and package cell line, package cell line 293TN Cell.Pack out while carrying the false type slow virus of the sgRNA and Cas9 of targeting DEAF1 genes；

(4) using the false type slow-virus infection aim cell, add after puromycin resistance screenings and further cultivate；

(5) monoclonal cell is spread, monoclonal cell is then collected, genomic DNA is extracted, using its genomic DNA as template amplification Include the genetic fragment of the target sequence, TA cloning and sequencings confirm that DEAF1 genes have been knocked and have obtained the thin of gene knockout Born of the same parents.

7. the cell strain according to claim 1 for knocking out DEAF1 genes is people's HT-29 cells；It is characterized in that：It is HT- The cell strain that DEAF1 gene delections or insertion nucleotide in 29 cells obtain, is any in following cell strain 3# and 5# Strain：Cell strain 3# is：The cleavage site of sgRNA is located at before areas sgRNA PAM on wild type DEAF1 genes at 7 bases, The second exon of DEAF1 genes is caused to lack 134 bases；Cell strain 5#：Be sgRNA cleavage site be located at wild Before the areas sgRNA PAM on type DEAF1 genes at 33 bases, the second exon of DEAF1 genes is caused to lack 160 alkali Base.