质粒名称: | pSECC-sgTom |
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出品公司: | addgene |
目录编号: | 138661 |
存储实验室: | Reuben Shaw |
载体骨架基本信息 |
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载体名称: | pSECC |
完整质粒大小: | 12046bp |
载体类型: | Lentiviral |
载体抗性: | 氨苄霉素 |
生长温度: | 30 ℃ |
宿主菌: | NEB Stable |
拷贝数: | 未知 |
插入基因信息 |
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插入基因名称: | sgTomato |
物种来源: | M. musculus (mouse) |
启动子: | hU6 |
克隆方法: | 限制性内切酶 |
5’克隆位点: | 未知(未破坏) |
文献引用方法 |
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材料和方法部分: | pSECC-sgTom was a gift from Reuben Shaw (Addgene plasmid # 138661 ; http://n2t.net/addgene:138661 ; RRID:Addgene_138661) |
参考文献部分: | The AMPK-Related Kinases SIK1 and SIK3 Mediate Key Tumor-Suppressive Effects of LKB1 in NSCLC. Hollstein PE, Eichner LJ, Brun SN, Kamireddy A, Svensson RU, Vera LI, Ross DS, Rymoff TJ, Hutchins A, Galvez HM, Williams AE, Shokhirev MN, Screaton RA, Berdeaux R, Shaw RJ. Cancer Discov. 2019 Nov;9(11):1606-1627. doi: 10.1158/2159-8290.CD-18-1261. Epub 2019 Jul 26. 10.1158/2159-8290.CD-18-1261 PubMed 31350328 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pSECC-sgTom 12046 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses a Tomato-targeting sgRNA, Cas9, and Cre-recombinase. ACCESSION . VERSION . KEYWORDS pSECC-sgTom SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12046) AUTHORS Hollstein PE, Eichner LJ, Brun SN, Kamireddy A, Svensson RU, Vera LI, Ross DS, Rymoff TJ, Hutchins A, Galvez HM, Williams AE, Shokhirev MN, Screaton RA, Berdeaux R, Shaw RJ TITLE The AMPK-Related Kinases SIK1 and SIK3 Mediate Key Tumor-Suppressive Effects of LKB1 in NSCLC. JOURNAL Cancer Discov. 2019 Nov;9(11):1606-1627. doi: 10.1158/2159-8290.CD-18-1261. Epub 2019 Jul 26. PUBMED 31350328 REFERENCE 2 (bases 1 to 12046) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..12046 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(46..65) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 237..616 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 618..816 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 771..791 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 834..1014 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1061..1186 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1683..1916 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2101..2145 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 2443..2560 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2660..2900 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2660..2680 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2831..2850 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 2929..3004 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 3052..3169 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 3268..3479 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 3524..3533 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3533..3556 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 3563..3583 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3608..7708 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 7709..7756 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 7772..7828 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 7832..7852 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7853..8881 /codon_start=1 /product="site-specific recombinase" /label=Cre /note="Cre is a site-specific recombinase from bacteriophage P1. Recombination occurs at loxP sequences." /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR YLAWSGHSARVGAARDMARAGVSIPGIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED GD" misc_feature 8934..9522 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(8987..9007) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(9405..9416) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(9525..9541) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9526..9542) /label=pBluescriptKS /note="For pBluescript vector" LTR 10051..10231 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(10293..10881) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(10373..10392) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(11052..11912) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 11675..11694 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(11913..12017) /gene="bla" /label=AmpR promoter ORIGIN 1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga 61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct 181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca 241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 781 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc 841 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 901 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 961 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 1021 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg 1081 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat 1141 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg 1201 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa 1261 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct 1321 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac 1381 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca 1441 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa 1501 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat 1561 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1621 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1681 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1741 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1801 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1861 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1921 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1981 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 2041 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 2101 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 2161 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 2221 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2281 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2341 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2401 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2461 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2521 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2581 cagagatcca gtttggttag taccgggccc gggtcgacta gtgatcagtg tgagggagtg 2641 taaagctggg tttcccagag agggcctatt tcccatgatt ccttcatatt tgcatatacg 2701 atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa agatattagt 2761 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg 2821 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt 2881 atatatcttg tggaaaggac gaaacaccgg ccacgagttc gagatcgagt tttagagcta 2941 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg 3001 gtgctttttt gaattctaga tcttgagaca aatggcagta ttcatccaca attttaaaag 3061 aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga 3121 catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta 3181 cagggacagc agagatccac tttggcgccg gcaaaccgct gttcctagga atcccgaggc 3241 cttcgagtgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga 3301 agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact 3361 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata 3421 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg 3481 tgtcgtgacg cgggacccga cattagcgct acagcttaag cgggccacca tggattacaa 3541 agacgatgac gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc 3601 agcagccgac aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc 3661 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga 3721 ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc 3781 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat 3841 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca 3901 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt 3961 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag 4021 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc 4081 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag 4141 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa 4201 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag 4261 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg 4321 aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc 4381 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct 4441 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc 4501 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc 4561 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt 4621 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta 4681 cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat 4741 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct 4801 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct 4861 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa 4921 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa 4981 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga 5041 ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga 5101 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac 5161 cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt 5221 cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt 5281 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga 5341 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa 5401 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat 5461 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta 5521 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg 5581 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat 5641 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga 5701 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag 5761 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca 5821 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat 5881 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga 5941 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca 6001 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg 6061 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga 6121 ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag 6181 aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat 6241 gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa 6301 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag 6361 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat 6421 gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa 6481 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa 6541 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa 6601 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg 6661 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta 6721 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa 6781 gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga 6841 ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga 6901 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct 6961 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt 7021 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag 7081 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat 7141 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc 7201 taagtactcc ctgttcgagc tggaaaacgg caggaagaga atgctggcct ctgccggcga 7261 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc 7321 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt 7381 ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag 7441 agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga 7501 taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg 7561 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac 7621 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg 7681 gatcgacctg tctcagctgg gaggcgacaa gcgtcctgct gctactaaga aagctggtca 7741 agctaagaaa aagaaagcta gcggcagcgg cgccaccaac ttcagcctgc tgaagcaggc 7801 cggcgacgtg gaggagaacc ccggccccat gcccaagaag aagaggaagg tgtccaattt 7861 actgaccgta caccaaaatt tgcctgcatt accggtcgat gcaacgagtg atgaggttcg 7921 caagaacctg atggacatgt tcagggatcg ccaggcgttt tctgagcata cctggaaaat 7981 gcttctgtcc gtttgccggt cgtgggcggc atggtgcaag ttgaataacc ggaaatggtt 8041 tcccgcagaa cctgaagatg ttcgcgatta tcttctatat cttcaggcgc gcggtctggc 8101 agtaaaaact atccagcaac atttgggcca gctaaacatg cttcatcgtc ggtccgggct 8161 gccacgacca agtgacagca atgctgtttc actggttatg cggcggatcc gaaaagaaaa 8221 cgttgatgcc ggtgaacgtg caaaacaggc tctagcgttc gaacgcactg atttcgacca 8281 ggttcgttca ctcatggaaa atagcgatcg ctgccaggat atacgtaatc tggcatttct 8341 ggggattgct tataacaccc tgttacgtat agccgaaatt gccaggatca gggttaaaga 8401 tatctcacgt actgacggtg ggagaatgtt aatccatatt ggcagaacga aaacgctggt 8461 tagcaccgca ggtgtagaga aggcacttag cctgggggta actaaactgg tcgagcgatg 8521 gatttccgta tctggtgtag ctgatgatcc gaataactac ctgttttgcc gggtcagaaa 8581 aaatggtgtt gccgcgccat ctgccaccag ccagctatca actcgcgccc tggaagggat 8641 ttttgaagca actcatcgat tgatttacgg cgctaaggat gactctggtc agagatacct 8701 ggcctggtct ggacacagtg cccgtgtcgg agccgcgcga gatatggccc gcgctggagt 8761 ttcaataccg gggatcatgc aagctggtgg ctggaccaat gtaaatattg tcatgaacta 8821 tatccgtaac ctggatagtg aaacaggggc aatggtgcgc ctgctggaag atggcgatta 8881 gaaacgttgt tgtttggggt tgaattactc ttctagaccg cggcgcgccc gataatcaac 8941 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 9001 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 9061 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg 9121 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 9181 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 9241 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 9301 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg 9361 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 9421 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc 9481 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgacctc 9541 gatcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct 9601 gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt cacacctcag 9661 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa 9721 aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg 9781 atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc 9841 agatatccac tgacctttgg atggtgctac aagctagtac cagttgagca agagaaggta 9901 gaagaagcca atgaaggaga gaacacccgc ttgttacacc ctgtgagcct gcatgggatg 9961 gatgacccgg agagagaagt attagagtgg aggtttgaca gccgcctagc atttcatcac 10021 atggcccgag agctgcatcc ggactgtact gggtctctct ggttagacca gatctgagcc 10081 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga 10141 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga 10201 cccttttagt cagtgtggaa aatctctagc agcatgtgag caaaaggcca gcaaaaggcc 10261 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 10321 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 10381 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 10441 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 10501 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 10561 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 10621 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 10681 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 10741 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 10801 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 10861 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 10921 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 10981 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 11041 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 11101 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 11161 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 11221 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 11281 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 11341 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 11401 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 11461 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 11521 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 11581 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 11641 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 11701 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 11761 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 11821 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 11881 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 11941 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 12001 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacg //