质粒名称: | pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT) |
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目录编号: | 79620 |
存储实验室: | Akihiko Kondo |
载体骨架基本信息 |
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载体名称: | pcDNA3.1 |
载体出品公司: | Invitrogen |
空载体大小: | 5428bp |
完整质粒大小: | 10414 bp |
筛选标记: | Neomycin (select with G418) |
载体抗性: | Ampicillin, 100 μg/mL |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | SpCas9 |
物种来源: | Streptococcus pyogenes |
基因突变: | D10A for nickase Cas9 |
启动子: | CMV |
标签/融合蛋白: |
PmCDA1 (C terminal on insert)
UGI (C terminal on insert)
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克隆方法: | Gibson Cloning |
5’测序引物 | ttcgctattacgccagctg |
3’测序引物 | GACAGGTCGGTCTTGACA |
文献引用方法 |
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材料和方法部分: | pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT) was a gift from Akihiko Kondo (Addgene plasmid # 79620 ; http://n2t.net/addgene:79620 ; RRID:Addgene_79620) |
参考文献部分: | Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A. Science. 2016 Aug 4. pii: aaf8729. 10.1126/science.aaf8729 PubMed 2749247 |
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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 pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT) 10419 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses nCas9-PmCDA1-UGI and gRNA(HPRT) in mammalian cells. ACCESSION . VERSION . KEYWORDS pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10419) AUTHORS Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A TITLE Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. JOURNAL Science. 2016 Aug 4. pii: aaf8729. PUBMED 27492474 REFERENCE 2 (bases 1 to 10419) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..10419 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(35..55) /label=T3 /note="T3 promoter, forward primer" promoter complement(35..53) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(74..90) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(74..90) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(87..109) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 98..114 /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)." promoter complement(122..152) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 167..188 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(305..322) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(476..1064) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(556..575) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1235..2095) /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 1858..1877 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2096..2200) /gene="bla" /label=AmpR promoter rep_origin complement(2226..2681) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2363..2384) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2575..2594 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2808..2830 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2822..2839 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2823..2839 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 2849..2867 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind 3027..3046 /label=H1 /note="Human H1 promoter, forward primer" misc_RNA 3136..3211 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 3226..3605 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 3606..3809 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 3760..3780 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" promoter 3854..3872 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 3901..3921 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3946..8049 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 8062..8082 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 8290..8355 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 8989..9009 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9016..9267 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 9268..9321 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 9328..10122 /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 GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(9382..9401) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 9992..10011 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 10296..10417 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(10333..10352) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 10387..10406 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" ORIGIN 1 ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct 61 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 121 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 181 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 241 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 301 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 361 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 421 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 481 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 541 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 601 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 661 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 721 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 781 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 841 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 901 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 961 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 1021 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 1081 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 1141 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 1201 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 1261 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 1321 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 1381 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 1441 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 1501 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 1561 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 1621 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 1681 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 1741 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 1801 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 1861 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 1921 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 1981 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 2041 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 2101 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 2161 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 2221 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 2281 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 2341 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 2401 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 2461 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 2521 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 2581 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 2641 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct 2701 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 2761 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 2821 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac 2881 cgggccaatt cgaacgctga cgtcatcaac ccgctccaag gaatcgcggg cccagtgtca 2941 ctaggcggga acacccagcg cgcgtgcgcc ctggcaggaa gatggctgtg agggacaggg 3001 gagtggcgcc ctgcaatatt tgcatgtcgc tatgtgttct gggaaatcac cataaacgtg 3061 aaatgtcttt ggatttggga atcttataag ttctgtatga ggaccacaga tccccccgag 3121 atgtcatgaa agagagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 3181 tcaacttgaa aaagtggcac cgagtcggtg ctttttttac gcgttgacat tgattattga 3241 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 3301 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 3361 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 3421 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 3481 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 3541 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 3601 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 3661 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 3721 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 3781 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact 3841 ggcttatcga aattaatacg actcactata gggagaccca agctggctag cgaaatggca 3901 ccgaagaaga agcgtaaagt cggaatccac ggagttcctg cggcaatgga caagaagtac 3961 tccattgggc tcgctatcgg cacaaacagc gtcggttggg ccgtcattac ggacgagtac 4021 aaggtgccga gcaaaaaatt caaagttctg ggcaataccg atcgccacag cataaagaag 4081 aacctcattg gcgccctcct gttcgactcc ggggagacgg ccgaagccac gcggctcaaa 4141 agaacagcac ggcgcagata tacccgcaga aagaatcgga tctgctacct gcaggagatc 4201 tttagtaatg agatggctaa ggtggatgac tctttcttcc ataggctgga ggagtccttt 4261 ttggtggagg aggataaaaa gcacgagcgc cacccaatct ttggcaatat cgtggacgag 4321 gtggcgtacc atgaaaagta cccaaccata tatcatctga ggaagaagct tgtagacagt 4381 actgataagg ctgacttgcg gttgatctat ctcgcgctgg cgcatatgat caaatttcgg 4441 ggacacttcc tcatcgaggg ggacctgaac ccagacaaca gcgatgtcga caaactcttt 4501 atccaactgg ttcagactta caatcagctt ttcgaagaga acccgatcaa cgcatccgga 4561 gttgacgcca aagcaatcct gagcgctagg ctgtccaaat cccggcggct cgaaaacctc 4621 atcgcacagc tccctgggga gaagaagaac ggcctgtttg gtaatcttat cgccctgtca 4681 ctcgggctga cccccaactt taaatctaac ttcgacctgg ccgaagatgc caagcttcaa 4741 ctgagcaaag acacctacga tgatgatctc gacaatctgc tggcccagat cggcgaccag 4801 tacgcagacc tttttttggc ggcaaagaac ctgtcagacg ccattctgct gagtgatatt 4861 ctgcgagtga acacggagat caccaaagct ccgctgagcg ctagtatgat caagcgctat 4921 gatgagcacc accaagactt gactttgctg aaggcccttg tcagacagca actgcctgag 4981 aagtacaagg aaattttctt cgatcagtct aaaaatggct acgccggata cattgacggc 5041 ggagcaagcc aggaggaatt ttacaaattt attaagccca tcttggaaaa aatggacggc 5101 accgaggagc tgctggtaaa gcttaacaga gaagatctgt tgcgcaaaca gcgcactttc 5161 gacaatggaa gcatccccca ccagattcac ctgggcgaac tgcacgctat cctcaggcgg 5221 caagaggatt tctacccctt tttgaaagat aacagggaaa agattgagaa aatcctcaca 5281 tttcggatac cctactatgt aggccccctc gcccggggaa attccagatt cgcgtggatg 5341 actcgcaaat cagaagagac catcactccc tggaacttcg aggaagtcgt ggataagggg 5401 gcctctgccc agtccttcat cgaaaggatg actaactttg ataaaaatct gcctaacgaa 5461 aaggtgcttc ctaaacactc tctgctgtac gagtacttca cagtttataa cgagctcacc 5521 aaggtcaaat acgtcacaga agggatgaga aagccagcat tcctgtctgg agagcagaag 5581 aaagctatcg tggacctcct cttcaagacg aaccggaaag ttaccgtgaa acagctcaaa 5641 gaagactatt tcaaaaagat tgaatgtttc gactctgttg aaatcagcgg agtggaggat 5701 cgcttcaacg catccctggg aacgtatcac gatctcctga aaatcattaa agacaaggac 5761 ttcctggaca atgaggagaa cgaggacatt cttgaggaca ttgtcctcac ccttacgttg 5821 tttgaagata gggagatgat tgaagaacgc ttgaaaactt acgctcatct cttcgacgac 5881 aaagtcatga aacagctcaa gaggcgccga tatacaggat gggggcggct gtcaagaaaa 5941 ctgatcaatg ggatccgaga caagcagagt ggaaagacaa tcctggattt tcttaagtcc 6001 gatggatttg ccaaccggaa cttcatgcag ttgatccatg atgactctct cacctttaag 6061 gaggacatcc agaaagcaca agtttctggc cagggggaca gtcttcacga gcacatcgct 6121 aatcttgcag gtagcccagc tatcaaaaag ggaatactgc agaccgttaa ggtcgtggat 6181 gaactcgtca aagtaatggg aaggcataag cccgagaata tcgttatcga gatggcccga 6241 gagaaccaaa ctacccagaa gggacagaag aacagtaggg aaaggatgaa gaggattgaa 6301 gagggtataa aagaactggg gtcccaaatc cttaaggaac acccagttga aaacacccag 6361 cttcagaatg agaagctcta cctgtactac ctgcagaacg gcagggacat gtacgtggat 6421 caggaactgg acatcaatcg gctctccgac tacgacgtgg atcatatcgt gccccagtct 6481 tttctcaaag atgattctat tgataataaa gtgttgacaa gatccgataa aaatagaggg 6541 aagagtgata acgtcccctc agaagaagtt gtcaagaaaa tgaaaaatta ttggcggcag 6601 ctgctgaacg ccaaactgat cacacaacgg aagttcgata atctgactaa ggctgaacga 6661 ggtggcctgt ctgagttgga taaagccggc ttcatcaaaa ggcagcttgt tgagacacgc 6721 cagatcacca agcacgtggc ccaaattctc gattcacgca tgaacaccaa gtacgatgaa 6781 aatgacaaac tgattcgaga ggtgaaagtt attactctga agtctaagct ggtctcagat 6841 ttcagaaagg actttcagtt ttataaggtg agagagatca acaattacca ccatgcgcat 6901 gatgcctacc tgaatgcagt ggtaggcact gcacttatca aaaaatatcc caagcttgaa 6961 tctgaatttg tttacggaga ctataaagtg tacgatgtta ggaaaatgat cgcaaagtct 7021 gagcaggaaa taggcaaggc caccgctaag tacttctttt acagcaatat tatgaatttt 7081 ttcaagaccg agattacact ggccaatgga gagattcgga agcgaccact tatcgaaaca 7141 aacggagaaa caggagaaat cgtgtgggac aagggtaggg atttcgcgac agtccggaag 7201 gtcctgtcca tgccgcaggt gaacatcgtt aaaaagaccg aagtacagac cggaggcttc 7261 tccaaggaaa gtatcctccc gaaaaggaac agcgacaagc tgatcgcacg caaaaaagat 7321 tgggacccca agaaatacgg cggattcgat tctcctacag tcgcttacag tgtactggtt 7381 gtggccaaag tggagaaagg gaagtctaaa aaactcaaaa gcgtcaagga actgctgggc 7441 atcacaatca tggagcgatc aagcttcgaa aaaaacccca tcgactttct cgaggcgaaa 7501 ggatataaag aggtcaaaaa agacctcatc attaagcttc ccaagtactc tctctttgag 7561 cttgaaaacg gccggaaacg aatgctcgct agtgcgggcg agctgcagaa aggtaacgag 7621 ctggcactgc cctctaaata cgttaatttc ttgtatctgg ccagccacta tgaaaagctc 7681 aaagggtctc ccgaagataa tgagcagaag cagctgttcg tggaacaaca caaacactac 7741 cttgatgaga tcatcgagca aataagcgaa ttctccaaaa gagtgatcct cgccgacgct 7801 aacctcgata aggtgctttc tgcttacaat aagcacaggg ataagcccat cagggagcag 7861 gcagaaaaca ttatccactt gtttactctg accaacttgg gcgcgcctgc agccttcaag 7921 tacttcgaca ccaccataga cagaaagcgg tacacctcta caaaggaggt cctggacgcc 7981 acactgattc atcagtcaat tacggggctc tatgaaacaa gaatcgacct ctctcagctc 8041 ggtggagaca gcagggctga ccccaagaag aagaggaagg tgggtggagg aggtaccggc 8101 ggtggaggct cagcagaata cgtacgagct ctgtttgact tcaatgggaa tgacgaggag 8161 gatctcccct ttaagaaggg cgatattctc cgcatcagag ataagcccga agaacaatgg 8221 tggaatgccg aggatagcga agggaaaagg ggcatgattc tggtgccata tgtggagaaa 8281 tattccggtg actacaaaga ccatgatggg gattacaaag accacgacat cgactacaaa 8341 gacgacgacg ataaatcagg gatgacagac gccgagtacg tgcgcattca tgagaaactg 8401 gatatttaca ccttcaagaa gcagttcttc aacaacaaga aatctgtgtc acaccgctgc 8461 tacgtgctgt ttgagttgaa gcgaaggggc gaaagaaggg cttgcttttg gggctatgcc 8521 gtcaacaagc cccaaagtgg caccgagaga ggaatacacg ctgagatatt cagtatccga 8581 aaggtggaag agtatcttcg ggataatcct gggcagttta cgatcaactg gtattccagc 8641 tggagtcctt gcgctgattg tgccgagaaa attctggaat ggtataatca ggaacttcgg 8701 ggaaacgggc acacattgaa aatctgggcc tgcaagctgt actacgagaa gaatgcccgg 8761 aaccagatag gactctggaa tctgagggac aatggtgtag gcctgaacgt gatggtttcc 8821 gagcactatc agtgttgtcg gaagattttc atccaaagct ctcataacca gctcaatgaa 8881 aaccgctggt tggagaaaac actgaaacgt gcggagaagt ggagatccga gctgagcatc 8941 atgatccagg tcaagattct gcataccact aagtctccag ccgttggtcc caagaagaaa 9001 agaaaagtcg gtaccatgac caacctttcc gacatcatag agaaggaaac aggcaaacag 9061 ttggtcatcc aagagtcgat actcatgctt cctgaagaag ttgaggaggt cattgggaat 9121 aagccggaaa gtgacattct cgtacacact gcgtatgatg agagcaccga tgagaacgtg 9181 atgctgctca cgtcagatgc cccagagtac aaaccctggg ctctggtgat tcaggactct 9241 aatggagaga acaagatcaa gatgctagaa ggcaggggaa gccttctgac ttgtggggat 9301 gtggaagaaa accctggtcc atctagaatg attgaacaag atggattgca cgcaggttct 9361 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc 9421 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc 9481 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc 9541 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg 9601 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag 9661 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc 9721 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt 9781 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc 9841 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc 9901 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg 9961 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag 10021 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg 10081 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg 10141 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct 10201 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc 10261 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg 10321 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 10381 ctagttgtgg tttgtccaaa ctcatcaatg tatcttagc //