质粒名称: | pHDE-35S-Cas9-mCherry |
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出品公司: | 爱迪基因 |
目录编号: | 78931 |
存储实验室: | Yunde Zhao |
载体骨架基本信息 |
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载体名称: | pHDE |
载体出品公司: | Zhao Lab |
载体抗性: | 大观霉素 |
空载体大小: | 8000 bp |
完整质粒大小: | -- |
载体修饰: | mCherry表达式盒35S-Cas9 |
载体类型: | 植物表达,CRISPR |
筛选标记: | 潮霉素;麦氏荧光 |
生长条件: | 37 ℃ |
插入基因信息 |
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克隆方法: | Gibson克隆 |
5' 测序引物 | CTGTGGTTGGCATGCACATAC |
文献引用方法 |
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材料和方法部分: | pHDE-35S-Cas9-mCherry was a gift from Yunde Zhao (Addgene plasmid # 78931 ; http://n2t.net/addgene:78931 ; RRID:Addgene_78931) |
参考文献部分: | An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. Gao X, Chen J, Dai X, Zhang D, Zhao Y. Plant Physiol. 2016 May 15. pii: pp.00663.2016. 10.1104/pp.16.00663 PubMed 27208253 |
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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 pHDE-35S-Cas9-mCherry 15443 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION For CRISPR/Cas9 mediated gene editing in Arabidopsis; provides a visual screen for Cas9-free plants. ACCESSION . VERSION . KEYWORDS pHDE-35S-Cas9-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15443) AUTHORS Gao X, Chen J, Dai X, Zhang D, Zhao Y TITLE An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. JOURNAL Plant Physiol. 2016 May 15. pii: pp.00663.2016. PUBMED 27208253 REFERENCE 2 (bases 1 to 15443) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15443 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 1..25 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" primer_bind complement(632..650) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 900..919 /label=mCherry-F /note="mCherry, forward primer" terminator 1203..1455 /label=MAS terminator /note="mannopine synthase terminator" promoter 1927..2106 /label=NOS promoter /note="nopaline synthase promoter" promoter 1959..2142 /function="NOS promoter" /label=NOS promoter /note="nopaline synthase promoter" terminator 3257..3509 /label=NOS terminator /note="nopaline synthase terminator and poly(A) signal" promoter 3642..3987 /label=CaMV 35S promoter /note="strong constitutive promoter from cauliflower mosaic virus" primer_bind 3930..3950 /label=35S promoter /note="CaMV 35S promoter, forward primer" CDS 3992..8095 /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 8108..8128 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(8640..8657) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(8640..8656) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8649..8671) /label=M13/pUC Forward /note="In lacZ gene" misc_feature 8878..8902 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" CDS 9430..10221 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" rep_origin 10465..11053 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 10954..10973 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 11207..11224 /label=L4440 /note="L4440 vector, forward primer" misc_feature 11239..11379 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(11305..11324) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 11465..11487 /label=pGEX 3' /note="pGEX vectors, reverse primer" rep_origin 11723..11917 /label=pVS1 oriV /note="origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" CDS complement(11983..13050) /codon_start=1 /product="replication protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /label=pVS1 RepA /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV MRYRNLIEGEASAGS" CDS complement(13484..14113) /codon_start=1 /product="stability protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /label=pVS1 StaA /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI" ORIGIN 1 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactagtgaa gtgaagcttg 61 gtctcaacta tgagacctaa gctggcacaa ctatatttcc aacatcacta gctaccatca 121 aaagattgac ttctcatctt actcgattga aaccaaatta acatagggtt tttatttaaa 181 taaaagttta accttctttt taaaaaattg ttcatagtgt catgtcagaa caagagctac 241 aaatcacaca tagcatgcat aagcggagct atgatgagtg gtattgtttt gttcgtcact 301 tgtcactctt ttccaacaca taatcccgac aacaacgtaa gagcatctct ctctctccac 361 acacactcat gcatgcatgc attcttacac gtgattgcca tgcaaatctc ctttctcacc 421 tataaataca aaccaaccct tcactacact cttcactcaa accaaaacaa gaaaacatac 481 acaaatagca aaacggtacc aacaatggat aacatggcca tcatcaagga gttcatgcgc 541 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc 601 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc 661 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg 721 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg 781 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg 841 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc 901 cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag 961 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac 1021 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac 1081 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag 1141 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtgagag 1201 ctcttggact cccatgttgg caaaggcaac caaacaaaca atgaatgatc cgctcctgca 1261 tatggggcgg tttgagtatt tcaactgcca tttgggctga attgaagaca tgctcctgtc 1321 agaaattccg tgatcttact caatattcag taatctcggc caatatccta aatgtgcgtg 1381 gctttatctg tctttgtatt gtttcatcaa ttcatgtaac gtttgctttt cttatgaatt 1441 ttcaaataaa ttatcgtatt gagaccgaat tctgtctaga cctaactgag taagctagct 1501 tgttcgagta ttatggcatt gggaaaactg tttttcttgt accatttgtt gtgcttgtaa 1561 tttactgtgt tttttattcg gttttcgcta tcgaactgtg aaatggaaat ggatggagaa 1621 gagttaatga atgatatggt ccttttgttc attctcaaat taatattatt tgttttttct 1681 cttatttgtt gtgtgttgaa tttgaaatta taagagatat gcaaacattt tgttttgagt 1741 aaaaatgtgt caaatcgtgg cctctaatga ccgaagttaa tatgaggagt aaaacactag 1801 atccccaaac aagcttgaaa ctgaaggcgg gaaacgacaa tctgatcatg agcggagaat 1861 taagggagtc acgttatgac ccccgccgat gacgcgggac aagccgtttt acgtttggaa 1921 ctgacagaac cgcaacgttg aaggagccac tcagccgcgg gtttctggag tttaatgagc 1981 taagcacata cgtcagaaac cattattgcg cgttcaaaag tcgcctaagg tcactatcag 2041 ctagcaaata tttcttgtca aaaatgctcc actgacgttc cataaattcc cctcggtatc 2101 caattagagt ctcatattca ctctcaatcc aaataatctg caccggatcc gctagaggat 2161 ctcgacctgc aagatcccgg ggggcaatga gatatgaaaa agcctgaact caccgcgacg 2221 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg 2281 gagggcgaag aatctcgtgc tttcagcttc gatataggag ggcgtggata tgtcctgcgg 2341 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg 2401 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat 2461 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc 2521 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag 2581 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat 2641 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc 2701 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc 2761 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc 2821 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc 2881 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc 2941 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt 3001 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg 3061 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc 3121 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga 3181 aaccgacgcc ccagcactcg tccgggatct tggaggtgat gtaacatgat cacaagctga 3241 tcccccgaat ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc 3301 tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat 3361 aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca 3421 attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc 3481 gcgcgcggtg tcatctatgt tactagatcg ggaattgatc ccccctcgac agcttgcatg 3541 ccagcttggg ctgcaggtcg acgcgtcaac atggtggagc acgacactct cgtctactcc 3601 aagaatatca aagatacagt ctcagaagac caaagggcta ttgagacttt tcaacaaagg 3661 gtaatatcgg gaaacctcct cggattccat tgcccagcta tctgtcactt catcaaaagg 3721 acagtagaaa aggaaggtgg cacctacaaa tgccatcatt gcgataaagg aaaggctatc 3781 gttcaagatg cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc 3841 gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgatatctcc 3901 actgacgtaa gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa 3961 ggaagttcat ttcatttgga gaggacacga tatggacaag aagtactcca ttgggctcga 4021 tatcggcaca aacagcgtcg gctgggccgt cattacggac gagtacaagg tgccgagcaa 4081 aaaattcaaa gttctgggca ataccgatcg ccacagcata aagaagaacc tcattggcgc 4141 cctcctgttc gactccgggg agacggccga agccacgcgg ctcaaaagaa cagcacggcg 4201 cagatatacc cgcagaaaga atcggatctg ctacctgcag gagatcttta gtaatgagat 4261 ggctaaggtg gatgactctt tcttccatag gctggaggag tcctttttgg tggaggagga 4321 taaaaagcac gagcgccacc caatctttgg caatatcgtg gacgaggtgg cgtaccatga 4381 aaagtaccca accatatatc atctgaggaa gaagcttgta gacagtactg ataaggctga 4441 cttgcggttg atctatctcg cgctggcgca tatgatcaaa tttcggggac acttcctcat 4501 cgagggggac ctgaacccag acaacagcga tgtcgacaaa ctctttatcc aactggttca 4561 gacttacaat cagcttttcg aagagaaccc gatcaacgca tccggagttg acgccaaagc 4621 aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa aacctcatcg cacagctccc 4681 tggggagaag aagaacggcc tgtttggtaa tcttatcgcc ctgtcactcg ggctgacccc 4741 caactttaaa tctaacttcg acctggccga agatgccaag cttcaactga gcaaagacac 4801 ctacgatgat gatctcgaca atctgctggc ccagatcggc gaccagtacg cagacctttt 4861 tttggcggca aagaacctgt cagacgccat tctgctgagt gatattctgc gagtgaacac 4921 ggagatcacc aaagctccgc tgagcgctag tatgatcaag cgctatgatg agcaccacca 4981 agacttgact ttgctgaagg cccttgtcag acagcaactg cctgagaagt acaaggaaat 5041 tttcttcgat cagtctaaaa atggctacgc cggatacatt gacggcggag caagccagga 5101 ggaattttac aaatttatta agcccatctt ggaaaaaatg gacggcaccg aggagctgct 5161 ggtaaagctt aacagagaag atctgttgcg caaacagcgc actttcgaca atggaagcat 5221 cccccaccag attcacctgg gcgaactgca cgctatcctc aggcggcaag aggatttcta 5281 cccctttttg aaagataaca gggaaaagat tgagaaaatc ctcacatttc ggatacccta 5341 ctatgtaggc cccctcgccc ggggaaattc cagattcgcg tggatgactc gcaaatcaga 5401 agagaccatc actccctgga acttcgagga agtcgtggat aagggggcct ctgcccagtc 5461 cttcatcgaa aggatgacta actttgataa aaatctgcct aacgaaaagg tgcttcctaa 5521 acactctctg ctgtacgagt acttcacagt ttataacgag ctcaccaagg tcaaatacgt 5581 cacagaaggg atgagaaagc cagcattcct gtctggagag cagaagaaag ctatcgtgga 5641 cctcctcttc aagacgaacc ggaaagttac cgtgaaacag ctcaaagaag actatttcaa 5701 aaagattgaa tgtttcgact ctgttgaaat cagcggagtg gaggatcgct tcaacgcatc 5761 cctgggaacg tatcacgatc tcctgaaaat cattaaagac aaggacttcc tggacaatga 5821 ggagaacgag gacattcttg aggacattgt cctcaccctt acgttgtttg aagataggga 5881 gatgattgaa gaacgcttga aaacttacgc tcatctcttc gacgacaaag tcatgaaaca 5941 gctcaagagg cgccgatata caggatgggg gcggctgtca agaaaactga tcaatgggat 6001 ccgagacaag cagagtggaa agacaatcct ggattttctt aagtccgatg gatttgccaa 6061 ccggaacttc atgcagttga tccatgatga ctctctcacc tttaaggagg acatccagaa 6121 agcacaagtt tctggccagg gggacagtct tcacgagcac atcgctaatc ttgcaggtag 6181 cccagctatc aaaaagggaa tactgcagac cgttaaggtc gtggatgaac tcgtcaaagt 6241 aatgggaagg cataagcccg agaatatcgt tatcgagatg gcccgagaga accaaactac 6301 ccagaaggga cagaagaaca gtagggaaag gatgaagagg attgaagagg gtataaaaga 6361 actggggtcc caaatcctta aggaacaccc agttgaaaac acccagcttc agaatgagaa 6421 gctctacctg tactacctgc agaacggcag ggacatgtac gtggatcagg aactggacat 6481 caatcggctc tccgactacg acgtggatca tatcgtgccc cagtcttttc tcaaagatga 6541 ttctattgat aataaagtgt tgacaagatc cgataaaaat agagggaaga gtgataacgt 6601 cccctcagaa gaagttgtca agaaaatgaa aaattattgg cggcagctgc tgaacgccaa 6661 actgatcaca caacggaagt tcgataatct gactaaggct gaacgaggtg gcctgtctga 6721 gttggataaa gccggcttca tcaaaaggca gcttgttgag acacgccaga tcaccaagca 6781 cgtggcccaa attctcgatt cacgcatgaa caccaagtac gatgaaaatg acaaactgat 6841 tcgagaggtg aaagttatta ctctgaagtc taagctggtc tcagatttca gaaaggactt 6901 tcagttttat aaggtgagag agatcaacaa ttaccaccat gcgcatgatg cctacctgaa 6961 tgcagtggta ggcactgcac ttatcaaaaa atatcccaag cttgaatctg aatttgttta 7021 cggagactat aaagtgtacg atgttaggaa aatgatcgca aagtctgagc aggaaatagg 7081 caaggccacc gctaagtact tcttttacag caatattatg aattttttca agaccgagat 7141 tacactggcc aatggagaga ttcggaagcg accacttatc gaaacaaacg gagaaacagg 7201 agaaatcgtg tgggacaagg gtagggattt cgcgacagtc cggaaggtcc tgtccatgcc 7261 gcaggtgaac atcgttaaaa agaccgaagt acagaccgga ggcttctcca aggaaagtat 7321 cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa aaagattggg accccaagaa 7381 atacggcgga ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg ccaaagtgga 7441 gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca caatcatgga 7501 gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag gcgaaaggat ataaagaggt 7561 caaaaaagac ctcatcatta agcttcccaa gtactctctc tttgagcttg aaaacggccg 7621 gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg cactgccctc 7681 taaatacgtt aatttcttgt atctggccag ccactatgaa aagctcaaag ggtctcccga 7741 agataatgag cagaagcagc tgttcgtgga acaacacaaa cactaccttg atgagatcat 7801 cgagcaaata agcgaattct ccaaaagagt gatcctcgcc gacgctaacc tcgataaggt 7861 gctttctgct tacaataagc acagggataa gcccatcagg gagcaggcag aaaacattat 7921 ccacttgttt actctgacca acttgggcgc gcctgcagcc ttcaagtact tcgacaccac 7981 catagacaga aagcggtaca cctctacaaa ggaggtcctg gacgccacac tgattcatca 8041 gtcaattacg gggctctatg aaacaagaat cgacctctct cagctcggtg gagacagcag 8101 ggctgacccc aagaagaaga ggaaggtgtg atcgatccag gcctcccagc tttcgtccgt 8161 atcatcggtt tcgacaacgt tcgtcaagtt caatgcatca gtttcattgc ccacacacca 8221 gaatcctact aagtttgagt attatggcat tggaaaagct gttttcttct atcatttgtt 8281 ctgcttgtaa tttactgtgt tctttcagtt tttgttttcg gacatcaaaa tgcaaatgga 8341 tggataagag ttaataaatg atatggtcct tttgttcatt ctcaaattat tattatctgt 8401 tgtttttact ttaatgggtt gaatttaagt aagaaaggaa ctaacagtgt gatattaagg 8461 tgcaatgtta gacatataaa acagtctttc acctctcttt ggttatgtct tgaattggtt 8521 tgtttcttca cttatctgtg taatcaagtt tactatgagt ctatgatcaa gtaattatgc 8581 aatcaagtta agtacagtat aggctttttg tgtcgagggg gtaccgagtc gaggaattca 8641 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 8701 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 8761 ccttcccaac agttgcgcag cctgaatggc gggtaccgag ctcgaattca attcggcgtt 8821 aattcagtac attaaaaacg tccgcaatgt gttattaagt tgtctaagcg tcaatttgtt 8881 tacaccacaa tatatcctgc caccagccag ccaacagctc cccgaccggc agctcggcac 8941 aaaatcacca ctcgatacag gcagcccatc agtccgggac ggcgtcagcg ggagagccgt 9001 tgtaaggcgg cagactttgc tcatgttacc gatgctattc ggaagaacgg caactaagct 9061 gccgggtttg aaacacggat gatctcgcgg agggtagcat gttgattgta acgatgacag 9121 agcgttgctg cctgtgatca attcgggcac gaacccagtg gacataagcc tcgttcggtt 9181 cgtaagctgt aatgcaagta gcgtaactgc cgtcacgcaa ctggtccaga accttgaccg 9241 aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttcttgttat gacatgtttt 9301 tttggggtac agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga 9361 tgtttgatgt tatggagcag caacgatgtt acgcagcagg gcagtcgccc taaaacaaag 9421 ttaaacatca tgggggaagc ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt 9481 ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg tacatttgta cggctccgca 9541 gtggatggcg gcctgaagcc acacagtgat attgatttgc tggttacggt gaccgtaagg 9601 cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc ggcttcccct 9661 ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt 9721 ccgtggcgtt atccagctaa gcgcgaactg caatttggag aatggcagcg caatgacatt 9781 cttgcaggta tcttcgagcc agccacgatc gacattgatc tggctatctt gctgacaaaa 9841 gcaagagaac atagcgttgc cttggtaggt ccagcggcgg aggaactctt tgatccggtt 9901 cctgaacagg atctatttga ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc 9961 gactgggctg gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg gtacagcgca 10021 gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga gcgcctgccg 10081 gcccagtatc agcccgtcat acttgaagct agacaggctt atcttggaca agaagaagat 10141 cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc actacgtgaa aggcgagatc 10201 accaaggtag tcggcaaata atgtctagct agaaattcgt tcaagccgac gccgcttcgc 10261 cggcgttaac tcaagcgatt agatgcacta agcacataat tgctcacagc caaactatca 10321 ggtcaagtct gcttttatta tttttaagcg tgcataataa gccctacaca aattgggaga 10381 tatatcatgc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 10441 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 10501 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 10561 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 10621 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 10681 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 10741 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 10801 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 10861 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 10921 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 10981 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 11041 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 11101 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 11161 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 11221 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 11281 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta 11341 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc 11401 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc 11461 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag 11521 ggtgccttga tgtgggcgcc ggcggtcgag tggcgacggc gcggcttgtc cgcgccctgg 11581 tagattgcct ggccgtaggc cagccatttt tgagcggcca gcggccgcga taggccgacg 11641 cgaagcggcg gggcgtaggg agcgcagcga ccgaagggta ggcgcttttt gcagctcttc 11701 ggctgtgcgc tggccagaca gttatgcaca ggccaggcgg gttttaagag ttttaataag 11761 ttttaaagag ttttaggcgg aaaaatcgcc ttttttctct tttatatcag tcacttacat 11821 gtgtgaccgg ttcccaatgt acggctttgg gttcccaatg tacgggttcc ggttcccaat 11881 gtacggcttt gggttcccaa tgtacgtgct atccacagga aagagacctt ttcgaccttt 11941 ttcccctgct agggcaattt gccctagcat ctgctccgta cattaggaac cggcggatgc 12001 ttcgccctcg atcaggttgc ggtagcgcat gactaggatc gggccagcct gccccgcctc 12061 ctccttcaaa tcgtactccg gcaggtcatt tgacccgatc agcttgcgca cggtgaaaca 12121 gaacttcttg aactctccgg cgctgccact gcgttcgtag atcgtcttga acaaccatct 12181 ggcttctgcc ttgcctgcgg cgcggcgtgc caggcggtag agaaaacggc cgatgccggg 12241 atcgatcaaa aagtaatcgg ggtgaaccgt cagcacgtcc gggttcttgc cttctgtgat 12301 ctcgcggtac atccaatcag ctagctcgat ctcgatgtac tccggccgcc cggtttcgct 12361 ctttacgatc ttgtagcggc taatcaaggc ttcaccctcg gataccgtca ccaggcggcc 12421 gttcttggcc ttcttcgtac gctgcatggc aacgtgcgtg gtgtttaacc gaatgcaggt 12481 ttctaccagg tcgtctttct gctttccgcc atcggctcgc cggcagaact tgagtacgtc 12541 cgcaacgtgt ggacggaaca cgcggccggg cttgtctccc ttcccttccc ggtatcggtt 12601 catggattcg gttagatggg aaaccgccat cagtaccagg tcgtaatccc acacactggc 12661 catgccggcc ggccctgcgg aaacctctac gtgcccgtct ggaagctcgt agcggatcac 12721 ctcgccagct cgtcggtcac gcttcgacag acggaaaacg gccacgtcca tgatgctgcg 12781 actatcgcgg gtgcccacgt catagagcat cggaacgaaa aaatctggtt gctcgtcgcc 12841 cttgggcggc ttcctaatcg acggcgcacc ggctgccggc ggttgccggg attctttgcg 12901 gattcgatca gcggccgctt gccacgattc accggggcgt gcttctgcct cgatgcgttg 12961 ccgctgggcg gcctgcgcgg ccttcaactt ctccaccagg tcatcaccca gcgccgcgcc 13021 gatttgtacc gggccggatg gtttgcgacc gtcacgccga ttcctcgggc ttgggggttc 13081 cagtgccatt gcagggccgg cagacaaccc agccgcttac gcctggccaa ccgcccgttc 13141 ctccacacat ggggcattcc acggcgtcgg tgcctggttg ttcttgattt tccatgccgc 13201 ctcctttagc cgctaaaatt catctactca tttattcatt tgctcattta ctctggtagc 13261 tgcgcgatgt attcagatag cagctcggta atggtcttgc cttggcgtac cgcgtacatc 13321 ttcagcttgg tgtgatcctc cgccggcaac tgaaagttga cccgcttcat ggctggcgtg 13381 tctgccaggc tggccaacgt tgcagccttg ctgctgcgtg cgctcggacg gccggcactt 13441 agcgtgtttg tgcttttgct cattttctct ttacctcatt aactcaaatg agttttgatt 13501 taatttcagc ggccagcgcc tggacctcgc gggcagcgtc gccctcgggt tctgattcaa 13561 gaacggttgt gccggcggcg gcagtgcctg ggtagctcac gcgctgcgtg atacgggact 13621 caagaatggg cagctcgtac ccggccagcg cctcggcaac ctcaccgccg atgcgcgtgc 13681 ctttgatcgc ccgcgacacg acaaaggccg cttgtagcct tccatccgtg acctcaatgc 13741 gctgcttaac cagctccacc aggtcggcgg tggcccatat gtcgtaaggg cttggctgca 13801 ccggaatcag cacgaagtcg gctgccttga tcgcggacac agccaagtcc gccgcctggg 13861 gcgctccgtc gatcactacg aagtcgcgcc ggccgatggc cttcacgtcg cggtcaatcg 13921 tcgggcggtc gatgccgaca acggttagcg gttgatcttc ccgcacggcc gcccaatcgc 13981 gggcactgcc ctggggatcg gaatcgacta acagaacatc ggccccggcg agttgcaggg 14041 cgcgggctag atgggttgcg atggtcgtct tgcctgaccc gcctttctgg ttaagtacag 14101 cgataacctt catgcgttcc ccttgcgtat ttgtttattt actcatcgca tcatatacgc 14161 agcgaccgca tgacgcaagc tgttttactc aaatacacat caccttttta gacggcggcg 14221 ctcggtttct tcagcggcca agctggccgg ccaggccgcc agcttggcat cagacaaacc 14281 ggccaggatt tcatgcagcc gcacggttga gacgtgcgcg ggcggctcga acacgtaccc 14341 ggccgcgatc atctccgcct cgatctcttc ggtaatgaaa aacggttcgt cctggccgtc 14401 ctggtgcggt ttcatgcttg ttcctcttgg cgttcattct cggcggccgc cagggcgtcg 14461 gcctcggtca atgcgtcctc acggaaggca ccgcgccgcc tggcctcggt gggcgtcact 14521 tcctcgctgc gctcaagtgc gcggtacagg gtcgagcgat gcacgccaag cagtgcagcc 14581 gcctctttca cggtgcggcc ttcctggtcg atcagctcgc gggcgtgcgc gatctgtgcc 14641 ggggtgaggg tagggcgggg gccaaacttc acgcctcggg ccttggcggc ctcgcgcccg 14701 ctccgggtgc ggtcgatgat tagggaacgc tcgaactcgg caatgccggc gaacacggtc 14761 aacaccatgc ggccggccgg cgtggtggtg tcggcccacg gctctgccag gctacgcagg 14821 cccgcgccgg cctcctggat gcgctcggca atgtccagta ggtcgcgggt gctgcgggcc 14881 aggcggtcta gcctggtcac tgtcacaacg tcgccagggc gtaggtggtc aagcatcctg 14941 gccagctccg ggcggtcgcg cctggtgccg gtgatcttct cggaaaacag cttggtgcag 15001 ccggccgcgt gcagttcggc ccgttggttg gtcaagtcct ggtcgtcggt gctgacgcgg 15061 gcatagccca gcaggccagc ggcggcgctc ttgttcatgg cgtaatgtct ccggttctag 15121 tcgcaagtat tctactttat gcgactaaaa cacgcgacaa gaaaacgcca ggaaaagggc 15181 agggcggcag cctgtcgcgt aacttaggac ttgtgcgaca tgtcgttttc agaagacggc 15241 tgcactgaac gtcagaagcc gactgcacta tagcagcgga ggggttggat caaagtactt 15301 taaagtactt taaagtactt taaagtactt tgatcccgag gggaaccctg tggttggcat 15361 gcacatacaa atggacgaac ggataaacct tttcacgccc ttttaaatat ccgattattc 15421 taataaacgc tcttttctct tag //