质粒名称: | p201N Cas9 |
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出品公司: | 爱迪基因 |
目录编号: | 59175 |
存储实验室: | Wayne Parrott |
载体骨架基本信息 |
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载体名称: | pPZP |
载体抗性: | 卡那霉素 |
空载体大小: | 6200 bp |
完整质粒大小: | 14349 bp |
载体修饰: | 利用aph基因对卡那霉素进行细菌筛选。 |
载体类型: | CRISPR |
筛选标记: | G418,卡那霉素 |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | Cas9,Cas9 human-codon optimized |
物种来源: | Synthetic |
插入大小: | 5265 bp |
启动子 | 2x35S |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | I-SceI(未销毁) |
3' 克隆位点: | I-SceI(未销毁) |
5' 测序引物 | ACATGCACCTAATTTCACTAGATGT |
3' 测序引物 | TGTAAAACGACGGCCAGT |
文献引用方法 |
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材料和方法部分: | p201N Cas9 was a gift from Wayne Parrott (Addgene plasmid # 59175 ; http://n2t.net/addgene:59175 ; RRID:Addgene_59175) |
参考文献部分: | Targeted genome modifications in soybean with CRISPR/Cas9. Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA. . BMC Biotechnology. 2015;15:16 10.1186/s12896-015-0131-2 |
买家导航
p201NCas9载体基本信息
p201NCas9载体图谱质粒图谱和多克隆位点信息
p201NCas9载体简介
p201NCas9载体序列
LOCUS p201N Cas9 14414 bp ds-DNA circular SYN 18-DEC-2019 DEFINITION Cas9 driven by double 35S, nptII for plant selection, I-PpoI site to accept gRNA from pUC gRNA Shuttle. ACCESSION . VERSION . KEYWORDS p201N Cas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14414) AUTHORS Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA. TITLE Targeted genome modifications in soybean with CRISPR/Cas9. JOURNAL BMC Biotechnology. 2015;15:16 REFERENCE 2 (bases 1 to 14414) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 18, 2019 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14414 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1217..1846 /codon_start=1 /product="stability protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /label=pVS1 StaA /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI" CDS 2275..3348 /codon_start=1 /product="replication protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /label=pVS1 RepA /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI LVMRYRNLIEGEASAGS" rep_origin 3414..3608 /label=pVS1 oriV /note="origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" primer_bind complement(3844..3866) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 3952..4092 /label=bom /note="basis of mobility region from pBR322" primer_bind 4007..4026 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(4107..4124) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4278..4866) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4358..4377) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4961..5776) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 5684..5703 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 5768..5787 /label=pENTR-R /note="pENTR vectors, reverse primer" misc_feature 6069..6093 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" protein_bind 6270..6291 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6306..6336 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6344..6360 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 6349..6371 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6368..6384 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6368..6384 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS complement(6906..7700) /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(7017..7036) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 7627..7646 /label=Neo-R /note="Neomycin resistance gene, reverse primer" promoter 8846..9522 /label=CaMV 35S promoter (enhanced) /note="cauliflower mosaic virus 35S promoter with a duplicated enhancer region" primer_bind 9459..9479 /label=35S promoter /note="CaMV 35S promoter, forward primer" CDS 9568..13671 /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 13684..13704 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 13771..14023 /label=NOS terminator /note="nopaline synthase terminator and poly(A) signal" primer_bind complement(14064..14081) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(14064..14080) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(14073..14095) /label=M13/pUC Forward /note="In lacZ gene" misc_feature 14276..14300 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" ORIGIN 1 agtactttaa agtactttaa agtactttaa agtactttga tccaacccct ccgctgctat 61 agtgcagtcg gcttctgacg ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag 121 tcctaagtta cgcgacaggc tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt 181 tttagtcgca taaagtagaa tacttgcgac tagaaccgga gacattacgc catgaacaag 241 agcgccgccg ctggcctgct gggctatgcc cgcgtcagca ccgacgacca ggacttgacc 301 aaccaacggg ccgaactgca cgcggccggc tgcaccaagc tgttttccga gaagatcacc 361 ggcaccaggc gcgaccgccc ggagctggcc aggatgcttg accacctacg ccctggcgac 421 gttgtgacag tgaccaggct agaccgcctg gcccgcagca cccgcgacct actggacatt 481 gccgagcgca tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac 541 accaccacgc cggccggccg catggtgttg accgtgttcg ccggcattgc cgagttcgag 601 cgttccctaa tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg 661 aagtttggcc cccgccctac cctcaccccg gcacagatcg cgcacgcccg cgagctgatc 721 gaccaggaag gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc 781 ctgtaccgcg cacttgagcg cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt 841 gccttccgtg aggacgcatt gaccgaggcc gacgccctgg cggccgccga gaatgaacgc 901 caagaggaac aagcatgaaa ccgcaccagg acggccagga cgaaccgttt ttcattaccg 961 aagagatcga ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct 1021 caaccgtgcg gctgcatgaa atcctggccg gtttgtctga tgccaagctg gcggcctggc 1081 cggccagctt ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg 1141 agtaaaacag cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta aataaacaaa 1201 tacgcaaggg gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa 1261 gacgaccatc gcaacccatc tagcccgcgc cctgcaactc gccggggccg atgttctgtt 1321 agtcgattcc gatccccagg gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc 1381 gctaaccgtt gtcggcatcg accgcccgac gattgaccgc gacgtgaagg ccatcggccg 1441 gcgcgacttc gtagtgatcg acggagcgcc ccaggcggcg gacttggctg tgtccgcgat 1501 caaggcagcc gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac 1561 cgccgacctg gtggagctgg ttaagcagcg cattgaggtc acggatggaa ggctacaagc 1621 ggcctttgtc gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc 1681 gctggccggg tacgagctgc ccattcttga gtcccgtatc acgcagcgcg tgagctaccc 1741 aggcactgcc gccgccggca caaccgttct tgaatcagaa cccgagggcg acgctgcccg 1801 cgaggtccag gcgctggccg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa 1861 gagaaaatga gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc acgcagcagc 1921 aaggctgcaa cgttggccag cctggcagac acgccagcca tgaagcgggt caactttcag 1981 ttgccggcgg aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt 2041 accgagctgc tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat 2101 gagtagatga attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac 2161 cgacgccgtg gaatgcccca tgtgtggagg aacgggcggt tggccaggcg taagcggctg 2221 ggttgtctgc cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgagc 2281 ggtcgcaaac catccggccc ggtacaaatc ggcgcggcgc tgggtgatga cctggtggag 2341 aagttgaagg ccgcgcaggc cgcccagcgg caacgcatcg aggcagaagc acgccccggt 2401 gaatcgtggc aagcggccgc tgatcgaatc cgcaaagaat cccggcaacc gccggcagcc 2461 ggtgcgccgt cgattaggaa gccgcccaag ggcgacgagc aaccagattt tttcgttccg 2521 atgctctatg acgtgggcac ccgcgatagt cgcagcatca tggacgtggc cgttttccgt 2581 ctgtcgaagc gtgaccgacg agctggcgag gtgatccgct acgagcttcc agacgggcac 2641 gtagaggttt ccgcagggcc ggccggcatg gccagtgtgt gggattacga cctggtactg 2701 atggcggttt cccatctaac cgaatccatg aaccgatacc gggaagggaa gggagacaag 2761 cccggccgcg tgttccgtcc acacgttgcg gacgtactca agttctgccg gcgagccgat 2821 ggcggaaagc agaaagacga cctggtagaa acctgcattc ggttaaacac cacgcacgtt 2881 gccatgcagc gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa 2941 gccttgatta gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag 3001 atcgagctag ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg 3061 acggttcacc ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg 3121 gcacgccgcg ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc 3181 agtggcagcg ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca 3241 aatgacctgc cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc 3301 atgcgctacc gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag 3361 atgctagggc aaattgccct agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat 3421 agcacgtaca ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattgggaac 3481 ccaaagccgt acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa 3541 ggcgattttt ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc 3601 tgtgcataac tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg 3661 tcgctgcgct ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca 3721 aaaatggctg gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca 3781 ctcgaccgcc ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga 3841 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg 3901 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat 3961 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag 4021 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 4081 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 4141 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 4201 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 4261 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 4321 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 4381 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 4441 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 4501 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 4561 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 4621 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 4681 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 4741 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 4801 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 4861 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 4921 cgttaaggga ttttggtcat gcaggatcat gaattaattc ttagaaaaac tcatcgagca 4981 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc 5041 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt 5101 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa 5161 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca 5221 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa 5281 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata 5341 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca 5401 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg 5461 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat 5521 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg 5581 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct 5641 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat 5701 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc 5761 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg 5821 ttcatgatct ggatcacagg cagcaacgct ctgtcatcgt tacaatcaac atgctaccct 5881 ccgcgagatc atccgtgttt caaacccggc agcttagttg ccgttcttcc gaatagcatc 5941 ggtaacatga gcaaagtctg ccgccttaca acggctctcc cgctgacgcc gtcccggact 6001 gatgggctgc ctgtatcgag tggtgatttt gtgccgagct gccggtcggg gagctgttgg 6061 ctggctggtg gcaggatata ttgtggtgta aacaaattga cgcttagaca acttaataac 6121 acattgcgga cgtttttaat gtactgaatt aacgccgaat tgctctagcc aatacgcaaa 6181 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 6241 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 6301 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 6361 tttcacacag gaaacagcta tgacatgatt acgaatcctt gactagaggg taggcgcggc 6421 ttaattaagc gcttaattaa gcgcttaatt aagcgcttaa taagccgcgc cgtaccccaa 6481 tagtctcgac agacacatag cacctaacat tattgcttaa atttactatt tttttactat 6541 attatactca acccaatgag cataaagact gtaaaatctc aaattcctga gaagcatatt 6601 tatcgatccc acagacttga tagttccata atccatacgc tgcagccaaa ttgctagtgt 6661 gttgaacatt taacacgtag agaactagaa aagatataaa actaagattg atatccaaaa 6721 tagacgagaa caataagcaa aaactcttag ttttgaaata aatcaacaat cccgagggtt 6781 gtcacataca tcaaaaacga aaatccatat agcaaaaaaa actctaaatt accgttcgac 6841 aaaaagagaa aactgatagg acatttgcta aacattaaaa tcaaaatttg gatcaagccg 6901 aattctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg 6961 gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata 7021 tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg 7081 atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg 7141 gtcacgacga gatcatcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct 7201 ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc 7261 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga 7321 tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca 7381 aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc 7441 gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat 7501 agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa 7561 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc 7621 tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc 7681 aatccatctt gttcaatcat ctcgagacgt accgcgcgat gcattcgaag atcgatcctt 7741 cgcctggagg agagaaatca gtggcgctgc ggcttttagg gtttctttgt tgatggaatg 7801 agagtgtaag ctctgccagt gccactttat tagggtttta caagcccttt tcttcgtaat 7861 tgggcctgac attttgtgcc acttgggcct ttagagatga aaatgtatat tgggcttaag 7921 ttgacttgaa ggataaatta gtttaggata ttacgttttt tatgagaatt ggtgtgtcgg 7981 atacatgtat atgatgcatt caaatatatg tattctagat acatttaagt ttagatacaa 8041 tctaaaatgt gtctttaatt acaggactgt aactaaaata cttaatgtaa gaagaatatt 8101 actcctttaa tagcttttga gtatatctag tctaacatct tttaaaaaag tctaatttct 8161 ttcatttatt tttcgagcaa tagcaaagtg cataattatt tttttcttct agaaattcag 8221 atttgtttct ctaaattttg agattctttt ctcaattttg tatgtctaga gaacaatgtg 8281 tatttttcac tctagttggt tgttactttg ttgaatgttc tgataaaagt atattgttat 8341 ttctgaagta gatataaacc ttcatttgga aattatacat aaatcaaaat cgttaattat 8401 ctagatcaag atatatgccc ttttcctaat gtatttgata catgcaccta atttcactag 8461 atgtatcttt tctatttttt aaattatgaa tagttaattt tttccatatg tgtatttgat 8521 acatacttca tgactttaaa aaattaatta tataccagat atatgtattt aaaatttgtt 8581 atgtatttaa agtatgtata tgattattcg atattaatct cttcgatgaa atttaaatcg 8641 ataactatgt gctttggatc gatctgccca ctagtgagtc gtattacaat tcatgactct 8701 cttaaggtag ccaaaatcga tatctaggga taacagggta atcgatcact agaattcggc 8761 gcgccgggcc caacatggtg gagcacgaca ctctcgtcta ctccaagaat atcaaagata 8821 cagtctcaga agaccaaagg gctattgaga cttttcaaca aagggtaata tcgggaaacc 8881 tcctcggatt ccattgccca gctatctgtc acttcatcaa aaggacagta gaaaaggaag 8941 gtggcaccta caaatgccat cattgcgata aaggaaaggc tatcgttcaa gatgcctctg 9001 ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg 9061 ttccaaccac gtcttcaaag caagtggatt gatgtgaaca tggtggagca cgacactctc 9121 gtctactcca agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt 9181 caacaaaggg taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc 9241 atcaaaagga cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga 9301 aaggctatcg ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg 9361 aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt 9421 gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca agacccttcc 9481 tctatataag gaagttcatt tcatttggag aggacacgct gaaatcacca gtctctctct 9541 acaaatctat ctctctcgac cgctagcatg gacaagaagt actccattgg gctcgatatc 9601 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa 9661 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc 9721 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga 9781 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct 9841 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa 9901 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag 9961 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg 10021 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag 10081 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact 10141 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc 10201 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg 10261 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac 10321 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac 10381 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg 10441 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag 10501 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac 10561 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc 10621 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa 10681 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta 10741 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc 10801 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc 10861 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat 10921 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag 10981 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc 11041 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac 11101 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca 11161 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc 11221 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag 11281 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg 11341 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag 11401 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg 11461 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc 11521 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga 11581 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg 11641 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca 11701 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca 11761 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg 11821 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag 11881 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg 11941 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc 12001 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat 12061 cggctctccg actacgacgt ggatcatatc gtgccccagt cttttctcaa agatgattct 12121 attgataata aagtgttgac aagatccgat aaaaatagag ggaagagtga taacgtcccc 12181 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg 12241 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg 12301 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg 12361 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga 12421 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag 12481 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca 12541 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga 12601 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag 12661 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca 12721 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa 12781 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag 12841 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc 12901 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac 12961 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa 13021 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga 13081 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa 13141 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa 13201 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa 13261 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat 13321 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag 13381 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt 13441 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac 13501 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata 13561 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca 13621 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct 13681 gaccccaaga agaagaggaa ggtgtgaccg cggatatctg cagaagctta cgcgtcgacg 13741 tctcgaggcc ggccgagctc gaatttcccc gatcgttcaa acatttggca ataaagtttc 13801 ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac 13861 gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg 13921 attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac 13981 taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt gatatctagg 14041 gataacaggg taataagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac 14101 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 14161 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatga 14221 gcttgagctt ggatcagatt gtcgtttccc gccttcagtt taaactatca gtgtttgaca 14281 ggatatattg gcgggtaaac ctaagagaaa agagcgttta ttagaataat cggatattta 14341 aaagggcgtg aaaaggttta tccgttcgtc catttgtatg tgcatgccaa ccacagggtt 14401 cccctcggga tcaa //