质粒名称: | pREDCas9 |
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出品公司: | 爱迪基因 |
目录编号: | 71541 |
存储实验室: | Tao Chen |
载体骨架基本信息 |
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载体名称: | pSC101ts |
载体出品公司: | -- |
载体抗性: | 大观霉素 |
空载体大小: | -- |
完整质粒大小: | -- |
载体修饰: | -- |
载体类型: | 细菌表达 |
筛选标记: | -- |
高拷贝/低拷贝: | 低拷贝 |
生长条件: | 30 ℃ |
插入基因信息 |
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插入基因名称: | Cas9 |
物种来源: | S. pyogenes |
插入大小: | -- |
基因突变: | -- |
GenBank编号 | -- |
启动子 | -- |
标签/融合蛋白: | -- |
克隆方法: | -- |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | -- |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | pREDCas9 was a gift from Tao Chen (Addgene plasmid # 71541 ; http://n2t.net/addgene:71541 ; RRID:Addgene_71541) |
参考文献部分: | Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Li Y, Lin Z, Huang C, Zhang Y, Wang Z, Tang YJ, Chen T, Zhao X. Metab Eng. 2015 Sep;31:13-21. doi: 10.1016/j.ymben.2015.06.006. Epub 2015 Jun 30. 10.1016/j.ymben.2015.06.006 PubMed 26141150 |
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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 pREDCas9 15460 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Constitutive expression of Cas9, inducible expression of the Red recombineering system, and inducible expression of the plasmid curing system for CRISPR mediated genome editing of E. coli.. ACCESSION . VERSION . KEYWORDS pREDCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15460) AUTHORS Li Y, Lin Z, Huang C, Zhang Y, Wang Z, Tang YJ, Chen T, Zhao X TITLE Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. JOURNAL Metab Eng. 2015 Sep;31:13-21. doi: 10.1016/j.ymben.2015.06.006. Epub 2015 Jun 30. PUBMED 26141150 REFERENCE 2 (bases 1 to 15460) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15460 /organism="synthetic DNA construct" /mol_type="other DNA" misc_RNA complement(370..448) /label=tracrRNA /note="trans-activating CRISPR RNA for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 744..4850 /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" misc_feature 4871..5002 /label=crRNA leader /note="crRNA leader sequence for the Streptococcus pyogenes CRISPR/Cas system" repeat_region 5003..5038 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" CDS complement(5248..6126) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /label=araC /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE EKVNDVAVKLS" promoter 6153..6437 /gene="araBAD" /label=araBAD promoter /note="promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction (Guzman et al., 1995)" primer_bind 6360..6379 /label=pBAD Forward /note="For vectors with E. coli araBAD promoter, forward primer" repeat_region 6584..6619 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" CDS 7397..8188 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAAYWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" CDS complement(8512..9192) /codon_start=1 /gene="exo" /product="5' to 3' double-stranded DNA exonuclease in the lambda Red system" /label=Exo /note="creates 3' single-stranded DNA tails" /translation="MTPDIILQRTGIDVRAVEQGEDAWHKLRLGVITASEVHNVIAKPR SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG EQWR" CDS complement(9189..9974) /codon_start=1 /gene="bet" /product="single-stranded DNA binding recombinase in the lambda Red system" /label=Beta /note="mediates pairing of a single-stranded DNA with a complementary target" /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA" CDS complement(9980..10396) /codon_start=1 /gene="gam" /product="inhibitor of the host RecBCD nuclease in the lambda Red system" /label=Gam /note="protects the double-stranded DNA substrate for recombination" /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV" primer_bind complement(10419..10441) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 10430..10446 /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(10454..10484) /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS complement(10610..11671) /codon_start=1 /gene="recA" /product="DNA repair protein from E. coli" /label=RecA /translation="MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGS LSLDIALGAGGLPMGRIVEIYGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIY ARKLGVDIDNLLCSQPDTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIGDS HMGLAARMMSQAMRKLAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASV RLDIRRIGAVKEGENVVGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVK EKLIEKAGAWYSYKGEKIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSV DDSEGVAETNEDF" CDS complement(12160..13242) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 13204..13223 /label=LacI-R /note="LacI, reverse primer" CDS complement(13428..14378) /codon_start=1 /gene="rep101" /product="RepA protein needed for replication with the pSC101 origin" /label=Rep101 /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI" rep_origin 14426..14648 /label=pSC101 ori /note="low-copy replication origin that requires the Rep101 protein" ORIGIN 1 ctgctcgcgc aggctgggtg ccaagctctc gggtaacatc aaggcccgat ccttggagcc 61 cttgccctcc cgcacgatga tcgtgccgtg atcgaaatcc agatccttga cccgcagttg 121 caaaccctca ctgatccgca tgcggatctt gctgtaggca taggcttggt tatgccggta 181 ctgccgggcc tcttgcggga ttacgaaatc atcctgtgga gcttagtagg tttagcaaga 241 tggcagcgcc taaatgtaga atgataaaag gattaagaga ttaatttccc taaaaatgat 301 aaaacaagcg ttttgaaagc gcttgttttt ttggtttgca gtcagagtag aatagaagta 361 tcaaaaaaag caccgactcg gtgccacttt ttcaagttga taacggacta gccttatttt 421 aacttgctat gctgttttga atggttccaa caagattatt ttataacttt tataacaaat 481 aatcaaggag aaattcaaag aaatttatca gccataaaac aatacttaat actatagaat 541 gataacaaaa taaactactt tttaaaagaa ttttgtgtta taatctattt attattaagt 601 attgggtaat attttttgaa gagatatttt gaaaaagaaa aattaaagca tattaaacta 661 atttcggagg tcattaaaac tattattgaa atcatcaaac tcattatgga tttaatttaa 721 actttttatt ttaggaggca aaaatggata agaaatactc aataggctta gatatcggca 781 caaatagcgt cggatgggcg gtgatcactg atgaatataa ggttccgtct aaaaagttca 841 aggttctggg aaatacagac cgccacagta tcaaaaaaaa tcttataggg gctcttttat 901 ttgacagtgg agagacagcg gaagcgactc gtctcaaacg gacagctcgt agaaggtata 961 cacgtcggaa gaatcgtatt tgttatctac aggagatttt ttcaaatgag atggcgaaag 1021 tagatgatag tttctttcat cgacttgaag agtctttttt ggtggaagaa gacaagaagc 1081 atgaacgtca tcctattttt ggaaatatag tagatgaagt tgcttatcat gagaaatatc 1141 caactatcta tcatctgcga aaaaaattgg tagattctac tgataaagcg gatttgcgct 1201 taatctattt ggccttagcg catatgatta agtttcgtgg tcattttttg attgagggag 1261 atttaaatcc tgataatagt gatgtggaca aactatttat ccagttggta caaacctaca 1321 atcaattatt tgaagaaaac cctattaacg caagtggagt agatgctaaa gcgattcttt 1381 ctgcacgatt gagtaaatca agacgattag aaaatctcat tgctcagctc cccggtgaga 1441 agaaaaatgg cttatttggg aatctcattg ctttgtcatt gggtttgacc cctaatttta 1501 aatcaaattt tgatttggca gaagatgcta aattacagct ttcaaaagat acttacgatg 1561 atgatttaga taatttattg gcgcaaattg gagatcaata tgctgatttg tttttggcag 1621 ctaagaattt atcagatgct attttacttt cagatatcct aagagtaaat actgaaataa 1681 ctaaggctcc cctatcagct tcaatgatta aacgctacga tgaacatcat caagacttga 1741 ctcttttaaa agctttagtt cgacaacaac ttccagaaaa gtataaagaa atcttttttg 1801 atcaatcaaa aaacggatat gcaggttata ttgatggggg agctagccaa gaagaatttt 1861 ataaatttat caaaccaatt ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac 1921 taaatcgtga agatttgctg cgcaagcaac ggacctttga caacggctct attccccatc 1981 aaattcactt gggtgagctg catgctattt tgagaagaca agaagacttt tatccatttt 2041 taaaagacaa tcgtgagaag attgaaaaaa tcttgacttt tcgaattcct tattatgttg 2101 gtccattggc gcgtggcaat agtcgttttg catggatgac tcggaagtct gaagaaacaa 2161 ttaccccatg gaattttgaa gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg 2221 aacgcatgac aaactttgat aaaaatcttc caaatgaaaa agtactacca aaacatagtt 2281 tgctttatga gtattttacg gtttataacg aattgacaaa ggtcaaatat gttactgaag 2341 gaatgcgaaa accagcattt ctttcaggtg aacagaagaa agccattgtt gatttactct 2401 tcaaaacaaa tcgaaaagta accgttaagc aattaaaaga agattatttc aaaaaaatag 2461 aatgttttga tagtgttgaa atttcaggag ttgaagatag atttaatgct tcattaggta 2521 cctaccatga tttgctaaaa attattaaag ataaagattt tttggataat gaagaaaatg 2581 aagatatctt agaggatatt gttttaacat tgaccttatt tgaagatagg gagatgattg 2641 aggaaagact taaaacatat gctcacctct ttgatgataa ggtgatgaaa cagcttaaac 2701 gtcgccgtta tactggttgg ggacgtttgt ctcgaaaatt gattaatggt attagggata 2761 agcaatctgg caaaacaata ttagattttt tgaaatcaga tggttttgcc aatcgcaatt 2821 ttatgcagct gatccatgat gatagtttga catttaaaga agacattcaa aaagcacaag 2881 tgtctggaca aggcgatagt ttacatgaac atattgcaaa tttagctggt agccctgcta 2941 ttaaaaaagg tattttacag actgtaaaag ttgttgatga attggtcaaa gtaatggggc 3001 ggcataagcc agaaaatatc gttattgaaa tggcacgtga aaatcagaca actcaaaagg 3061 gccagaaaaa ttcgcgagag cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa 3121 gtcagattct taaagagcat cctgttgaaa atactcaatt gcaaaatgaa aagctctatc 3181 tctattatct ccaaaatgga agagacatgt atgtggacca agaattagat attaatcgtt 3241 taagtgatta tgatgtcgat cacattgttc cacaaagttt ccttaaagac gattcaatag 3301 acaataaggt cttaacgcgt tctgataaaa atcgtggtaa atcggataac gttccaagtg 3361 aagaagtagt caaaaagatg aaaaactatt ggagacaact tctaaacgcc aagttaatca 3421 ctcaacgtaa gtttgataat ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata 3481 aagctggttt tatcaaacgc caattggttg aaactcgcca aatcactaag catgtggcac 3541 aaattttgga tagtcgcatg aatactaaat acgatgaaaa tgataaactt attcgagagg 3601 ttaaagtgat taccttaaaa tctaaattag tttctgactt ccgaaaagat ttccaattct 3661 ataaagtacg tgagattaac aattaccatc atgcccatga tgcgtatcta aatgccgtcg 3721 ttggaactgc tttgattaag aaatatccaa aacttgaatc ggagtttgtc tatggtgatt 3781 ataaagttta tgatgttcgt aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa 3841 ccgcaaaata tttcttttac tctaatatca tgaacttctt caaaacagaa attacacttg 3901 caaatggaga gattcgcaaa cgccctctaa tcgaaactaa tggggaaact ggagaaattg 3961 tctgggataa agggcgagat tttgccacag tgcgcaaagt attgtccatg ccccaagtca 4021 atattgtcaa gaaaacagaa gtacagacag gcggattctc caaggagtca attttaccaa 4081 aaagaaattc ggacaagctt attgctcgta aaaaagactg ggatccaaaa aaatatggtg 4141 gttttgatag tccaacggta gcttattcag tcctagtggt tgctaaggtg gaaaaaggga 4201 aatcgaagaa gttaaaatcc gttaaagagt tactagggat cacaattatg gaaagaagtt 4261 cctttgaaaa aaatccgatt gactttttag aagctaaagg atataaggaa gttaaaaaag 4321 acttaatcat taaactacct aaatatagtc tttttgagtt agaaaacggt cgtaaacgga 4381 tgctggctag tgccggagaa ttacaaaaag gaaatgagct ggctctgcca agcaaatatg 4441 tgaatttttt atatttagct agtcattatg aaaagttgaa gggtagtcca gaagataacg 4501 aacaaaaaca attgtttgtg gagcagcata agcattattt agatgagatt attgagcaaa 4561 tcagtgaatt ttctaagcgt gttattttag cagatgccaa tttagataaa gttcttagtg 4621 catataacaa acatagagac aaaccaatac gtgaacaagc agaaaatatt attcatttat 4681 ttacgttgac gaatcttgga gctcccgctg cttttaaata ttttgataca acaattgatc 4741 gtaaacgata tacgtctaca aaagaagttt tagatgccac tcttatccat caatccatca 4801 ctggtcttta tgaaacacgc attgatttga gtcagctagg aggtgactga agtatatttt 4861 agatgaagat tatttcttaa taactaaaaa tatggtataa tactcttaat aaatgcagta 4921 atacaggggc ttttcaagac tgaagtctag ctgagacaaa tagtgcgatt acgaaatttt 4981 ttagacaaaa atagtctacg aggttttaga gctatgctgt tttgaatggt cccaaaactg 5041 cagcgcaata gttggcgaag taatcgcaac atccgcatta aaatctagcg agggctttac 5101 taagctccgc aaaaaacccc gcccctgaca gggcggggtt ttttcgcgcc cgatccttgg 5161 agcccttgcc ctcccgcacg atgatcgtgc cgtgatcgaa atccagatcc ttgacccgca 5221 gttgcaaacc ctcactgatc cgcatgctta tgacaacttg acggctacat cattcacttt 5281 ttcttcacaa ccggcacgga actcgctcgg gctggccccg gtgcattttt taaatacccg 5341 cgagaaatag agttgatcgt caaaaccaac attgcgaccg acggtggcga taggcatccg 5401 ggtggtgctc aaaagcagct tcgcctggct gatacgttgg tcctcgcgcc agcttaagac 5461 gctaatccct aactgctggc ggaaaagatg tgacagacgc gacggcgaca agcaaacatg 5521 ctgtgcgacg ctggcgatat caaaattgct gtctgccagg tgatcgctga tgtactgaca 5581 agcctcgcgt acccgattat ccatcggtgg atggagcgac tcgttaatcg cttccatgcg 5641 ccgcagtaac aattgctcaa gcagatttat cgccagcagc tccgaatagc gcccttcccc 5701 ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa tgcggctggt gcgcttcatc 5761 cgggcgaaag aaccccgtat tggcaaatat tgacggccag ttaagccatt catgccagta 5821 ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg cgagcctccg gatgacgacc 5881 gtagtgatga atctctcctg gcgggaacag caaaatatca cccggtcggc aaacaaattc 5941 tcgtccctga tttttcacca ccccctgacc gcgaatggtg agattgagaa tataaccttt 6001 cattcccagc ggtcggtcga taaaaaaatc gagataaccg ttggcctcaa tcggcgttaa 6061 acccgccacc agatgggcat taaacgagta tcccggcagc aggggatcat tttgcgcttc 6121 agccatactt ttcatactcc cgccattcag agaagaaacc aattgtccat attgcatcag 6181 acattgccgt cactgcgtct tttactggct cttctcgcta accaaaccgg taaccccgct 6241 tattaaaagc attctgtaac aaagcgggac caaagccatg acaaaaacgc gtaacaaaag 6301 tgtctataat cacggcagaa aagtccacat tgattatttg cacggcgtca cactttgcta 6361 tgccatagca tttttatcca taagattagc ggatcctacc tgacgctttt tatcgcaact 6421 ctctactgtt tctccatgta ggtcgttcgc tccaagcgtt ttagagctag aaatagcaag 6481 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accagacccc gttgatgata 6541 ccgctgcctt actgggtgca ttagccagtc tgaatgacct cgagttttag agctatgctg 6601 ttttgaatgg tcccaaaact tcagcacact gagacttgtt gagttccatg ttttagagct 6661 atgctgtttt gaatggactc cattcaacat tgccgatgat aacttgagaa agagggttaa 6721 taccagcagt cggatacctt cctattcttt ctgttaaagc gttttcatgt tataataggc 6781 aaaagaagag tagtgtgatc gtccattccg acgtcgagcc gttccataca gaagctgggc 6841 gaacaaacga tgctcgcctt ccagaaaacc gaggatgcga accacttcat ccggggtcag 6901 caccaccggc aagcgccgcg acggccgagg tcttccgatc tcctgaagcc agggcagatc 6961 cgtgcacagc accttgccgt agaagaacag caaggccgcc aatgcctgac gatgcgtgga 7021 gaccgaaacc ttgcgctcgt tcgccagcca ggacagaaat gcctcgactt cgctgctgcc 7081 caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc agtggacata 7141 agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca actggtccag 7201 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga 7261 ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt 7321 gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa 7381 aacaaagtta aacatcatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga 7441 ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg 7501 ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac 7561 cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc 7621 ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga 7681 catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa 7741 tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct 7801 gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga 7861 tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc 7921 gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta 7981 cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gcctactggg caatggagcg 8041 cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga 8101 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg 8161 cgagatcacc aaggtagtcg gcaaataatg tctaacaatt cgttcaagcc gacgccgctt 8221 cgcggcgcgg cttaactcaa gcgttagatg cactaagcac ataattgctc acagccaaac 8281 tatcaggtca agtctgcttt tattattttt aagcgtgcat aataagccct acacaaattg 8341 ggagatatat catgaaaggc tggctttttc ttgttatcgc aatagttggc gaagtaatcg 8401 caacatccgc attaaaatct agcgagggct ttactaagct cctgcagaga tctgaattcc 8461 ctagagagac gaaagtgatt gcgcctaccc ggatattatc gtgaggatgc gtcatcgcca 8521 ttgctcccca aatacaaaac caatttcagc cagtgcctcg tccatttttt cgatgaactc 8581 cggcacgatc tcgtcaaaac tcgccatgta cttttcatcc cgctcaatca cgacataatg 8641 caggccttca cgcttcatac gcgggtcata gttggcaaag taccaggcat tttttcgcgt 8701 cacccacatg ctgtactgca cctgggccat gtaagctgac tttatggcct cgaaaccacc 8761 gagccggaac ttcatgaaat cccgggaggt aaacgggcat ttcagttcaa ggccgttgcc 8821 gtcactgcat aaaccatcgg gagagcaggc ggtacgcata ctttcgtcgc gatagatgat 8881 cggggattca gtaacattca cgccggaagt gaattcaaac agggttctgg cgtcgttctc 8941 gtactgtttt ccccaggcca gtgctttagc gttaacttcc ggagccacac cggtgcaaac 9001 ctcagcaagc agggtgtgga agtaggacat tttcatgtca ggccacttct ttccggagcg 9061 gggttttgct atcacgttgt gaacttctga agcggtgatg acgccgagcc gtaatttgtg 9121 ccacgcatct tccccctgtt cgacagctct cacatcgatc ccggtacgct gcaggataat 9181 gtccggtgtc atgctgccac cttctgctct gcggctttct gtttcaggaa tccaagagct 9241 tttactgctt cggcctgtgt cagttctgac gatgcacgaa tgtcgcggcg aaatatctgg 9301 gaacagagcg gcaataagtc gtcatcccat gttttatcca gggcgatcag cagagtgtta 9361 atctcctgca tggtttcatc gttaaccgga gtgatgtcgc gttccggctg acgttctgca 9421 gtgtatgcag tattttcgac aatgcgctcg gcttcatcct tgtcatagat accagcaaat 9481 ccgaaggcca gacgggcaca ctgaatcatg gctttatgac gtaacatccg tttgggatgc 9541 gactgccacg gccccgtgat ttctctgcct tcgcgagttt tgaatggttc gcggcggcat 9601 tcatccatcc attcggtaac gcagatcgga tgattacggt ccttgcggta aatccggcat 9661 gtacaggatt cattgtcctg ctcaaagtcc atgccatcaa actgctggtt ttcattgatg 9721 atgcgggacc agccatcaac gcccaccacc ggaacgatgc cattctgctt atcaggaaag 9781 gcgtaaattt ctttcgtcca cggattaagg ccgtactggt tggcaacgat cagtaatgcg 9841 atgaactgcg catcgctggc atcaccttta aatgccgtct ggcgaagagt ggtgatcagt 9901 tcctgtgggt cgacagaatc catgccgaca cgttcagcca gcttcccagc cagcgttgcg 9961 agtgcagtac tcattcgttt tatacctctg aatcaatatc aacctggtgg tgagcaatgg 10021 tttcaaccat gtaccggatg tgttctgcca tgcgctcctg aaactcaaca tcgtcatcaa 10081 acgcacgggt aatggatttt ttgctggccc cgtggcgttg caaatgatcg atgcatagcg 10141 attcaaacag gtgctggggc aggccttttt ccatgtcgtc tgccagttct gcctctttct 10201 cttcacgggc gagctgctgg tagtgacgcg cccagctctg agcctcaaga cgatcctgaa 10261 tgtaataagc gttcatggct gaactcctga aatagctgtg aaaatatcgc ccgcgaaatg 10321 ccgggctgat taggaaaaca ggaaaggggg ttagtgaatg cttttgcttg atctcagttt 10381 cagtattaat atccattttt tataagcgtc gactgtttcc tgtgtgaaat tgttatccgc 10441 tcacaattcc acacattata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 10501 gagtgagaat tcggatctcg acgggatgtt gattctgtca tggcatatcc ttacaactta 10561 aaaaagcaaa agggccgcag atgcgaccct tgtgtatcaa acaagacgat taaaaatctt 10621 cgttagtttc tgctacgcct tcgctatcat ctacagagaa atccggcgtt gagttcgggt 10681 tgctcagcag caactcacgt actttcttct cgatctcttt cgcggtttcc gggttatctt 10741 tcagccaggc agtcgcattc gctttaccct gaccgatctt ctcacctttg tagctgtacc 10801 acgcgcctgc tttctcgatc agcttctctt ttacgcccag gtcaaccagt tcgccgtaga 10861 agttgatacc ttcgccgtag aggatctgga attcagcctg tttaaacggc gcagcgattt 10921 tgttcttcac cactttcacg cgggtttcgc tacccaccac gttttcgccc tctttcaccg 10981 cgccgatacg acggatgtcg agacgaacag aggcgtagaa tttcagcgcg ttaccaccgg 11041 tagtggtttc cgggttaccg aacatcacac caattttcat acggatctgg ttgatgaaga 11101 tcagcagcgt gttggactgc ttcaggttac ccgccagctt acgcatcgcc tggctcatca 11161 tacgtgccgc aaggcccatg tgagagtcgc cgatttcgcc ttcgatttcc gctttcggcg 11221 tcagtgccgc cacggagtca acgacgataa cgtctactgc gccagaacgc gccagggcgt 11281 cacagatttc cagtgcctgc tcgccggtgt ccggctggga gcacagcagg ttgtcgatat 11341 cgacgcccag tttacgtgcg tagattgggt ccagcgcgtg ttcagcatcg ataaacgcac 11401 aggttttacc ttcacgctgc gctgcggcga tcacctgcag cgtcagcgtg gttttaccgg 11461 aagattccgg tccgtagatt tcgacgatac ggcccatcgg cagaccacct gccccaagcg 11521 cgatatccag tgaaagcgaa ccggtagaga tggtttccac atccatggaa cggtcttcac 11581 ccaggcgcat gatggagcct ttaccaaatt gtttctcaat ctggcccagt gctgccgcca 11641 acgctttctg tttgttttcg tcgatagcca tttttactcc tgtcatgccg ggtaataccg 11701 gatagtcaat atgttctgtt gaagcaatta tactgtatgc tcatacagta tcaagtgttt 11761 tgtagaaatt gttgccacaa ggtctgcaat gcatacgcag tagcctgacg acgcaccgca 11821 tcacggtcgc cgctgaagca tcccgccggg taatgccttc accgcgggca gtggcaaaag 11881 caaaccagac ggtgccgaca ggcttctcta gatccgccta cctttcacga gttgcgcagt 11941 ttgtctgcaa gactctatga gaagcagata agcgataagt ttgctcaaca tcttctcggg 12001 cataagtcgg acaccatggc atcacagtat cgtgatgaca gaggcaggga gtgggacaaa 12061 attgaaatca aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg 12121 ataagcaatg ccaagcttgg cactggctga tcagctagct cactgcccgc tttccagtcg 12181 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 12241 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc 12301 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag 12361 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc 12421 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg 12481 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc 12541 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc 12601 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg 12661 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac 12721 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg 12781 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc 12841 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag 12901 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac 12961 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc 13021 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg 13081 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg 13141 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac 13201 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg 13261 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt cgatgctagc 13321 ccatgggtat ggacagtttt ccctttgata tgtaacggtg aacagttgtt ctacttttgt 13381 ttgttagtct tgatgcttca ctgatagata caagagccat aagaacctca gatccttccg 13441 tatttagcca gtatgttctc tagtgtggtt cgttgttttt gcgtgagcca tgagaacgaa 13501 ccattgagat catacttact ttgcatgtca ctcaaaaatt ttgcctcaaa actggtgagc 13561 tgaatttttg cagttaaagc atcgtgtagt gtttttctta gtccgttacg taggtaggaa 13621 tctgatgtaa tggttgttgg tattttgtca ccattcattt ttatctggtt gttctcaagt 13681 tcggttacga gatccatttg tctatctagt tcaacttgga aaatcaacgt atcagtcggg 13741 cggcctcgct tatcaaccac caatttcata ttgctgtaag tgtttaaatc tttacttatt 13801 ggtttcaaaa cccattggtt aagcctttta aactcatggt agttattttc aagcattaac 13861 atgaacttaa attcatcaag gctaatctct atatttgcct tgtgagtttt cttttgtgtt 13921 agttctttta ataaccactc ataaatcctc atagagtatt tgttttcaaa agacttaaca 13981 tgttccagat tatattttat gaattttttt aactggaaaa gataaggcaa tatctcttca 14041 ctaaaaacta attctaattt ttcgcttgag aacttggcat agtttgtcca ctggaaaatc 14101 tcaaagcctt taaccaaagg attcctgatt tccacagttc tcgtcatcag ctctctggtt 14161 gctttagcta atacaccata agcattttcc ctactgatgt tcatcatctg agcgtattgg 14221 ttataagtga acgataccgt ccgttctttc cttgtagggt tttcaatcgt ggggttgagt 14281 agtgccacac agcataaaat tagcttggtt tcatgctccg ttaagtcata gcgactaatc 14341 gctagttcat ttgctttgaa aacaactaat tcagacatac atctcaattg gtctaggtga 14401 ttttaatcac tataccaatt gagatgggct agtcaatgat aattactagt ccttttcctt 14461 tgagttgtgg gtatctgtaa attctgctag acctttgctg gaaaacttgt aaattctgct 14521 agaccctctg taaattccgc tagacctttg tgtgtttttt ttgtttatat tcaagtggtt 14581 ataatttata gaataaagaa agaataaaaa aagataaaaa gaatagatcc cagccctgtg 14641 tataactcac tactttagtc agttccgcag tattacaaaa ggatgtcgca aacgctgttt 14701 gctcctctac aaaacagacc ttaaaaccct aaaggcttaa gtagcaccct cgcaagctcg 14761 gttgcggccg caatcgggca aatcgctgaa tattcctttt gtctccgacc atcaggcacc 14821 tgagtcgctg tctttttcgt gacattcagt tcgctgcgct cacggctctg gcagtgaatg 14881 ggggtaaatg gcactacagg cgccttttat ggattcatgc aaggaaacta cccataatac 14941 aagaaaagcc cgtcacgggc ttctcagggc gttttatggc gggtctgcta tgtggtgcta 15001 tctgactttt tgctgttcag cagttcctgc cctctgattt tccagtctga ccacttcgga 15061 ttatcccgtg acaggtcatt cagactggct aatgcaccca gtaaggcagc ggtatcatca 15121 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 15181 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 15241 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 15301 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 15361 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 15421 tcaccggctc cagatttatc agcaataaac cagccagccg //