质粒名称: | pFC333 |
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出品公司: | Addgene |
目录编号: | 87844 |
存储实验室: | Uffe Mortensen |
载体骨架基本信息 |
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载体名称: | custom |
完整质粒大小: | 15667 bp |
载体类型: | 细菌表达,CRISPR;真菌表达 |
筛选标记: | Zeocin ; bleomycin, phleomycin |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
插入基因信息 |
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插入基因名称: | Cas9 |
物种来源: | 合成的黑曲霉合成基因,密码子优化 |
插入大小: | 4131 bp |
GenBank编号 | KT031983 |
启动子: | Aspergillus nidulans tef1 promoter |
标签/融合蛋白: | SV40 NLS(插入件上的C端子) |
5’测序引物 | CTTCTCTGCTCAGCACCTCTACG |
文献引用方法 |
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材料和方法部分: | pFC333 was a gift from Uffe Mortensen (Addgene plasmid # 87844 ; http://n2t.net/addgene:87844 ; RRID:Addgene_87844) |
参考文献部分: | A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. Nodvig CS, Nielsen JB, Kogle ME, Mortensen UH. PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. PONE-D-15-11561 [pii] PubMed 26177455 |
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载体基本信息
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载体序列
LOCUS pFC333 15668 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AMA1 plasmid with Aspergillus optimized Cas9 and ble selection marker. ACCESSION . VERSION . KEYWORDS pFC333 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15668) AUTHORS Nodvig CS, Nielsen JB, Kogle ME, Mortensen UH TITLE A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. JOURNAL PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. PUBMED 26177455 REFERENCE 2 (bases 1 to 15668) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15668 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind complement(63..96) /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" primer_bind complement(566..583) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(566..582) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(681..698) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(852..1440) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(932..951) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1562..2422) /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 2185..2204 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2423..2514) /gene="bla" /label=AmpR promoter primer_bind 2556..2575 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2581..2600 /label=pENTR-R /note="pENTR vectors, reverse primer" protein_bind 2807..2840 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" rep_origin 3153..8403 /label=AMA1 /note="replication origin from Aspergillus nidulans (Aleksenko and Clutterbuck, 1996)" CDS 9575..13678 /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 13682..13702 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 14528..14902 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" ORIGIN 1 aggtaaatcc ccactaccgc attaagacct cagcgcggcc gcaaatttaa ataaaatgaa 61 gtgaagttcc tatactttct agagaatagg aacttctata gtgagtcgaa taagggcgac 121 acaaaattta ttctaaatgc ataataaata ctgataacat cttatagttt gtattatatt 181 ttgtattatc gttgacatgt ataattttga tatcaaaaac tgattttccc tttattattt 241 tcgagattta ttttcttaat tctctttaac aaactagaaa tattgtatat acaaaaaatc 301 ataaataata gatgaatagt ttaattatag gtgttcatca atcaaaaaag caacgtatct 361 tatttaaagt gcgttgcttt tttctcattt ataaggttaa ataattctca tatatcaagc 421 aaagtgacag gcgcccttaa atattctgac aaatgctctt tccctaaact ccccccataa 481 aaaaacccgc cgaagcgggt ttttacgtta tttgcggatt aacgattact cgttatcaga 541 accgcccagg gggcccgagc ttaagactgg ccgtcgtttt acaacacaga aagagtttgt 601 agaaacgcaa aaaggccatc cgtcaggggc cttctgctta gtttgatgcc tggcagttcc 661 ctactctcgc cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 721 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 781 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 841 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 901 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 961 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 1021 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 1081 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 1141 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 1201 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 1261 aagtggtggg ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 1321 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 1381 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 1441 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgacgc gcgcgtaact 1501 cacgttaagg gattttggtc atgagcttgc gccgtcccgt caagtcagcg taatgctctg 1561 cttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 1621 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 1681 ccagcgctgc gatgataccg cgagaaccac gctcaccggc tccggattta tcagcaataa 1741 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 1801 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 1861 acgttgttgc catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 1921 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 1981 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 2041 tcatggttat ggcagcgcta cataattctc ttactgtcat gccatccgta agatgctttt 2101 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 2161 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 2221 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 2281 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 2341 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 2401 cacggaaatg ttgaatactc atattcttcc tttttcaata ttattgaagc atttatcagg 2461 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 2521 tcagtgttac aaccaattaa ccaattctga acattatcgc gagcccattt atacctgaat 2581 atggctcata acaccccttg tttgcctggc ggcagtagcg cggtggtccc acctgacccc 2641 atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggac tccccatgcg 2701 agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt 2761 tcgcccgggc taattatggg gtgtcgccct tattcgactc tatagtgaag ttcctattct 2821 ctagaaagta taggaacttc tgaagtgggg atttaaatgc ggccgcgctg agggtttaat 2881 cgacgaagca gctgacggcc agtgccaagc ttaacgcgta ccgggcccag tatatgttcc 2941 gcagatgact ggagctctgc catacgtgcc ctctcaagca ccatttgttc catctacaga 3001 gactagtcac caactagtct atcaagactc acagggtaca ttgctgagac caactgacca 3061 gaggcagggt agcggattga cggctccatc tccttcactt acaaggtcta ttgaaagccc 3121 tttagcatca ccaagcggag aatagattgt taagcttatt ttttgtatac tgttttgtga 3181 tagcacgaag tttttccacg gtatcttgtt aaaaatatat atttgtggcg ggcttaccta 3241 catcaaatta ataagagact aattataaac taaacacaca agcaagctac tttagggtaa 3301 aagtttataa atgcttttga cgtataaacg ttgcttgtat ttattattac aattaaaggt 3361 ggatagaaaa cctagagact agttagaaac taatctcagg tttgcgttaa actaaatcag 3421 agcccgagag gttaacagaa cctagaaggg gactagatat ccgggtaggg aaacaaaaaa 3481 aaaaaacaag acagccacat attagggaga ctagttagaa gctagttcca ggactaggaa 3541 aataaaagac aatgatacca cagtctagtt gacaactaga tagattctag attgaggcca 3601 aagtctctga gatccaggtt agttgcaact aatactagtt agtatctagt ctcctataac 3661 tctgaagcta gaataactta ctactattat cctcaccact gttcagctgc gcaaacggag 3721 tgattgcaag gtgttcagag actagttatt gactagtcag tgactagcaa taactaacaa 3781 ggtattaacc taccatgtct gccatcaccc tgcacttcct cgggctcagc agccttttcc 3841 tcctcatttt catgctcatt ttccttgttt aagactgtga ctagtcaaag actagtccag 3901 aaccacaaag gagaaatgtc ttaccacttt cttcattgct tgtctctttt gcattatcca 3961 tgtctgcaac tagttagagt ctagttagtg actagtccga cgaggacttg cttgtctccg 4021 gattgttgga ggaactctcc agggcctcaa gatccacaac agagccttct agaagactgg 4081 tcaataacta gttggtcttt gtctgagtct gacttacgag gttgcatact cgctcccttt 4141 gcctcgtcaa tcgatgagaa aaagcgccaa aactcgcaat atggctttga accacacggt 4201 gctgagacta gttagaatct agtcccaaac tagcttggat agcttacctt tgccctttgc 4261 gttgcgacag gtcttgcagg gtatggttcc tttctcacca gctgatttag ctgccttgct 4321 accctcacgg cggatctgca taaagagtgg ctagaggtta taaattagca ctgatcctag 4381 gtacggggct gaatgtaact tgccyttcct ttctcatcgc gcggcaagac aggcttgctc 4441 aaattcctac cagtcacagg ggtatgcacg gcgtacggac cacttgaact agtcacagat 4501 tagttagcaa ctagtctgca ttgaatggct gtacttacgg gccctcgcca ttgtcctgat 4561 catttccagc ttcaccctcg ttgctgcaaa gtagttagtg actagtcaag gactagttga 4621 aatgggagaa gaaactcacg aattctcgac acccttagta ttgtggtcct tggacttggt 4681 gctgctatat attagctaat acactagtta gactcacaga aacttacgca gctcgcttgc 4741 gcttcttggt aggagtcggg gttgggagaa cagtgccttc aaacaagcct tcataccatg 4801 ctacttgact agtcagggac tagtcaccaa gtaatctaga taggacttgc ctttggcctc 4861 catcagttcc ttcatagtgg gaggtccatt gtgcaatgta aactccatgc cgtgggagtt 4921 cttgtccttc aagtgcttga ccaatatgtt tctgttggca gagggaacct gtcaactagt 4981 taataactag tcagaaacta gtatagcagt agactcactg tacgcttgag gcatcccttc 5041 actcggcagt agacttcata tggatggata tcaggcacgc cattgtcgtc ctgtggacta 5101 gtcagtaact aggcttaaag ctagtcgggt cggcttacta tcttgaaatc cggcagcgta 5161 agctccccgt ccttaactgc ctcgagatag tgacagtact ctggggactt tcggagatcg 5221 ttatcgcgaa tgctcggcat actaatcgtt gactagtctt ggactagtcc cgagcaaaaa 5281 ggattggagg aggaggagga aggtgagagt gagacaaaga gcgaaataag agcttcaaag 5341 gctatctcta agcagtatga aggttaagta tctagttctt gactagattt aaaagagatt 5401 tcgactagtt atgtacctgg agtttggata taggaatgtg ttgtggtaac gaaatgtaag 5461 ggggaggaaa gaaaaagtcg gtcaagaggt aactctaagt cggccattcc tttttgggag 5521 gcgctaacca taaacggcat ggtcgactta gagttagctc agggaattta gggagttatc 5581 tgcgaccacc gaggaacggc ggaatgccaa agaatcccga tggagctcta gctggcggtt 5641 gacaacccca ccttttggcg tttctgcggc gttgcaggcg ggactggata cttcgtagaa 5701 ccagaaaggc aaggcagaac gcgctcagca agagtgttgg aagtgatagc atgatgtgcc 5761 ttgttaacta ggtcaaaatc tgcagtatgc ttgatgttat ccgaagtgtg agagaggaag 5821 gtccaaacat acacgattgg gagagggcct aggtataaga gtttttgagt agaacgcatg 5881 tgagcccagc catctcgagg agattaaaca cgggccggca tttgatggct atgttagtac 5941 cccaatggaa acggtgagag tccagtggtc gcagataact ccctaaattc cctgagctaa 6001 ctctaagtcg accatgccgt ttatggttag cgcctcccaa aaaggaatgg ccgacttaga 6061 gttacctctt gaccgacttt ttctttcctc ccccttacat ttcgttacca caacacattc 6121 ctatatccaa actccaggta cataactagt cgaaatctct tttaaatcta gtcaagaact 6181 agatacttaa ccttcatact gcttagagat agcctttgaa gctcttattt cgctctttgt 6241 ctcactctca ccttcctcct cctcctccaa tcctttttgc tcgggactag tccaagacta 6301 gtcaacgatt agtatgccga gcattcgcga taacgatctc cgaaagtccc cagagtactg 6361 tcactatctc gaggcagtta aggacgggga gcttacgctg ccggatttca agatagtaag 6421 ccgacccgac tagctttaag cctagttact gactagtcca caggacgaca atggcgtgcc 6481 tgatatccat ccatatgaag tctactgccg agtgaaggga tgcctcaagc gtacagtgag 6541 tctactgcta tactagtttc tgactagtta ttaactagtt gacaggttcc ctctgccaac 6601 agaaacatat tggtcaagca cttgaaggac aagaactccc acggcatgga gtttacattg 6661 cacaatggac ctcccactat gaaggaactg atggaggcca aaggcaagtc ctatctagat 6721 tacttggtga ctagtccctg actagtcaag tagcatggta tgaaggcttg tttgaaggca 6781 ctgttctccc aaccccgact cctaccaaga agcgcaagcg agctgcgtaa gtttctgtga 6841 gtctaactag tgtattagct aatatatagc agcaccaagt ccaaggacca caatactaag 6901 ggtgtcgaga attcgtgagt ttcttctccc atttcaacta gtccttgact agtcactaac 6961 tactttgcag caacgagggt gaagctggaa atgatcagga caatggcgag ggcccgtaag 7021 tacagccatt caatgcagac tagttgctaa ctaatctgtg actagttcaa gtggtccgta 7081 cgccgtgcat acccctgtga ctggtaggaa tttgagcaag cctgtcttgc cgcgcgatga 7141 gaaaggaarg gcaagttaca ttcagccccg tacctaggat cagtgctaat ttataacctc 7201 tagccactct ttatgcagat ccgccgtgag ggtagcaagg cagctaaatc agctggtgag 7261 aaaggaacca taccctgcaa gacctgtcgc aacgcaaagg gcaaaggtaa gctatccaag 7321 ctagtttggg actagattct aactagtctc agcaccgtgt ggttcaaagc catattgcga 7381 gttttggcgc tttttctcat cgattgacga ggcaaaggga gcgagtatgc aacctcgtaa 7441 gtcagactca gacaaagacc aactagttat tgaccagtct tctagaaggc tctgttgtgg 7501 atcttgaggc cctggagagt tcctccaaca atccggagac aagcaagtcc tcgtcggact 7561 agtcactaac tagactctaa ctagttgcag acatggataa tgcaaaagag acaagcaatg 7621 aagaaagtgg taagacattt ctcctttgtg gttctggact agtctttgac tagtcacagt 7681 cttaaacaag gaaaatgagc atgaaaatga ggaggaaaag gctgctgagc ccgaggaagt 7741 gcagggtgat ggcagacatg gtaggttaat accttgttag ttattgctag tcactgacta 7801 gtcaataact agtctctgaa caccttgcaa tcactccgtt tgcgcagctg aacagtggtg 7861 aggataatag tagtaagtta ttctagcttc agagttatag gagactagat actaactagt 7921 attagttgca actaacctgg atctcagaga ctttggcctc aatctagaat ctatctagtt 7981 gtcaactaga ctgtggtatc attgtctttt attttcctag tcctggaact agcttctaac 8041 tagtctccct aatatgtggc tgtcttgttt tttttttttt tgtttcccta cccggatatc 8101 tagtcccctt ctaggttctg ttaacctctc gggctctgat ttagtttaac gcaaacctga 8161 gattagtttc taactagtct ctaggttttc tatccacctt taattgtaat aataaataca 8221 agcaacgttt atacgtcaaa agcatttata aacttttacc ctaaagtagc ttgcttgtgt 8281 gtttagttta taattagtct cttattaatt tgatgtaggt aagcccgcca caaatatata 8341 tttttaacaa gataccgtgg aaaaacttcg tgctatcaca aaacagtata caaaaaataa 8401 gcttaacaat ctattctccg cttggtgatg ctaaagggct ttcaatagac cttgtaagtg 8461 aaggagatgg agccgtcaat ccgctaccct gcctctggtc agttggtctc agcaatgtac 8521 cctgtgagtc ttgatagact agttggtgac tagtctctgt agatggaaca aatggtgctt 8581 gagagggcac gtatggcaga gctccagtca tctgcggaac atatactggg cccgggaaga 8641 tctcatggtc atagctgttt ccgctgaggg tttaattaag acctcagccg agacagcaga 8701 atcaccgccc aagttaagcc tttgtgctga tcatgctctc gaacgggcca agttcgggaa 8761 aagcaaagga gcgtttagtg aggggcaatt tgactcacct cccaggcaac agatgagggg 8821 ggcaaaaaga aagaaatttt cgtgagtcaa tatggattcc gagcatcatt ttcttgcggt 8881 ctatcttgct acgtatgttg atcttgacgc tgtggatcaa gcaacgccac tcgctcgctc 8941 catcgcaggc tggtcgcaga caaattaaaa ggcggcaaac tcgtacagcc gcggggttgt 9001 ccgctgcaaa gtacagagtg ataaaagccg ccatgcgacc atcaacgcgt tgatgcccag 9061 ctttttcgat ccgagaatcc accgtagagg cgatagcaag taaagaaaag ctaaacaaaa 9121 aaaaatttct gcccctaagc catgaaaacg agatggggtg gagcagaacc aaggaaagag 9181 tcgcgctggg ctgccgttcc ggaaggtgtt gtaaaggctc gacgcccaag gtgggagtct 9241 aggagaagaa tttgcatcgg gagtggggcg ggttacccct ccatatccaa tgacagatat 9301 ctaccagcca agggtttgag cccgcccgct tagtcgtcgt cctcgcttgc ccctccataa 9361 aaggatttcc cctccccctc ccacaaaatt ttctttccct tcctctcctt gtccgcttca 9421 gtacgtatat cttcccttcc ctcgcttctc tcctccatcc ttctttcatc catctcctgc 9481 taacttctct gctcagcacc tctacgcatt actagccgta gtatctgagc acttctccct 9541 tttatattcc acaaaacata acacaacctt caccatggac aagaagtata gcatcgggct 9601 ggacattgga acgaactcgg ttggttgggc tgtgattacg gacgaataca aggtgccatc 9661 caagaagttt aaggtcctgg gaaacaccga ccgtcactca atcaagaaga atctcattgg 9721 agccctgctc ttcgatagtg gggagaccgc cgaagctact cgactgaagc gaacggctcg 9781 ccggcgttat acacgacgca agaatcgcat ctgctacctc caggagattt tcagcaacga 9841 aatggctaag gttgatgact cattctttca tcgactcgaa gaaagtttct tggtcgagga 9901 ggataagaag cacgagcgcc atccgatctt tggtaacatt gtggatgagg ttgcctatca 9961 cgaaaagtac ccaactatct atcatcttcg taagaagctg gtcgatagca cggacaaggc 10021 tgatttgcga cttatctacc tggcactcgc gcacatgatt aagttccgcg gccattttct 10081 tatcgagggt gacctgaacc ccgataattc tgacgttgat aagctcttca tccagttggt 10141 ccaaacctac aatcagctgt ttgaggaaaa ccctattaat gcatctggcg tggacgccaa 10201 ggctatcctt tcggcgcgcc tgtctaagtc gcggcgtttg gagaacctta tcgcacaact 10261 ccccggcgaa aagaagaacg gcctcttcgg taatttgatt gcgttgtcac ttggtctgac 10321 tcctaacttc aagagtaatt ttgacctggc agaggatgcg aagctccagt tgtctaagga 10381 tacgtatgat gacgatctcg acaacttgct tgcccaaatc ggtgaccagt acgctgatct 10441 tttcctggcc gctaagaatc tctcagatgc aatcctgctc agtgacattt tgcgggtcaa 10501 caccgagatt actaaggccc ccctgtcagc tagtatgatc aagcggtatg atgagcacca 10561 tcaggacctc accttgctta aggccctcgt gcgtcagcaa ttgcctgaga agtataagga 10621 aatcttcttt gaccaatcca agaacggata cgcagggtat attgatggcg gtgcgagcca 10681 ggaggaattc tacaagttta tcaagccgat tttggagaag atggacggca ctgaggaact 10741 gctcgtcaag ctgaatcgcg aagatttgct tcgtaagcaa cgaacgttcg acaacggctc 10801 catcccgcac cagattcatc tgggcgagct ccacgccatc cttcgacgcc aggaagattt 10861 ctacccattt ctgaaggaca accgtgagaa gatcgaaaag attcttacat tccgaatccc 10921 ctactatgtg ggacctttgg cccgtgggaa ttcccgattt gcttggatga cccgaaagag 10981 cgaggaaacc atcactccgt ggaacttcga ggaagtcgtg gacaagggtg catccgcgca 11041 gagcttcatt gagcggatga ccaattttga taagaacctt ccgaatgaaa aggtcctgcc 11101 aaagcattcg ctgctctacg agtatttcac cgtgtataac gaactgacta aggtcaagta 11161 cgtgacggag ggaatgcgga agccagcctt cctctcaggg gaacaaaaga aggctatcgt 11221 cgatttgctt tttaagacca atcgtaaagt gactgttaag cagctgaagg aggattattt 11281 caagaagatt gaatgtttcg actccgtcga gatcagcggc gtggaagatc gctttaacgc 11341 ttccctcggt acctaccacg acctgctcaa gatcattaag gacaaggatt tcctcgataa 11401 cgaggaaaat gaggacatct tggaagatat tgtcctcacg ttgacacttt ttgaggaccg 11461 cgaaatgatc gaggaacggc tcaagacata tgcccatttg ttcgacgata aggtgatgaa 11521 gcagctgaag cggcgtcgat acaccggatg gggtcgcctt agccggaagc tgatcaacgg 11581 cattcgagat aagcaatctg gtaagactat cttggatttc cttaagtcgg acggcttcgc 11641 caaccgcaat tttatgcagc ttattcacga cgattccctg acgttcaagg aggacatcca 11701 gaaggcacaa gtctcaggac aaggggattc cctgcacgag catatcgcca acctggctgg 11761 atccccggcg atcaagaagg ggattcttca gaccgtcaag gttgtcgacg agctggtcaa 11821 ggtgatgggc cgtcataagc cagaaaacat cgtgattgag atggcccgag aaaatcagac 11881 cactcaaaag ggtcagaaga acagccgcga gcggatgaag cggatcgagg aaggcattaa 11941 ggaacttggt tctcagatcc tgaaggagca ccctgttgaa aacacacagc tccaaaatga 12001 gaagctgtat ctctactatt tgcaaaatgg acgcgacatg tacgtcgatc aggagctcga 12061 cattaaccgg ttgtcggact acgatgttga ccatatcgtc ccgcaatcct tccttaagga 12121 cgatagcatt gataacaagg tgctgactcg ctcagataag aaccggggca agtccgacaa 12181 tgttccaagc gaggaagtgg ttaagaagat gaagaactac tggcgccaat tgcttaatgc 12241 caagctcatc acacagcgca agtttgacaa cttgaccaag gccgagcggg gagggctgag 12301 tgaactcgat aaggctggct tcatcaagcg tcaactcgtg gagacgcgac agatcacaaa 12361 gcacgttgct cagattctgg actcccggat gaacacaaag tacgacgaga atgataagct 12421 catccgtgaa gttaaggtca ttaccctcaa gtctaagttg gtgtcggatt tccgcaagga 12481 cttccaattt tataaggttc gggagatcaa caattatcac catgcacatg atgcgtacct 12541 caacgcagtc gtgggaactg cgctcatcaa gaagtatccc aagttggagt ccgaattcgt 12601 ctacggggat tataaggttt acgacgtccg caagatgatc gccaagagtg agcaggaaat 12661 tggcaaggcc acggctaagt atttctttta ctccaacatc atgaatttct ttaagacgga 12721 gatcacactc gccaatggag aaatccgtaa gcgacctttg attgagacca acggcgagac 12781 tggtgaaatc gtttgggata aggggcgcga cttcgctacc gtgcggaagg ttctgagcat 12841 gccgcaagtc aatatcgtca agaaaaccga ggtgcagaca ggcggtttct ctaaggaatc 12901 gattcttcca aagcgtaact ctgacaagct gatcgctcga aagaaggatt gggaccccaa 12961 gaagtatgga gggttcgatt ctcctacagt ggcatactcg gttctcgttg tcgcgaaggt 13021 tgagaaggga aagtctaaga agctgaagtc ggtcaaggaa ctgctcggga tcaccattat 13081 ggagcgctcc agcttcgaaa agaatcccat cgactttctc gaggccaagg gctataagga 13141 agtcaagaag gatcttatca ttaagctgcc taagtactct ttgttcgagc ttgaaaacgg 13201 tcgaaagcga atgctcgcat cggcaggaga gttgcagaag gggaatgaat tggcacttcc 13261 ctcaaagtac gtgaacttcc tgtatctcgc gtcccactac gagaagctga agggtagccc 13321 tgaggacaac gaacagaagc aactttttgt tgagcaacac aagcattatc tggatgagat 13381 cattgaacag atttcagagt tcagtaagcg cgtcatcctc gccgatgcta atctcgacaa 13441 ggtgttgtcg gcctacaaca agcaccgtga caagccgatc cgagagcagg ctgaaaatat 13501 cattcatctg ttcaccctca ctaacttggg agcaccagca gcgttcaagt attttgatac 13561 gacaatcgac cgtaagcgat acacgtccac aaaggaggtg cttgatgcga ccctgattca 13621 tcaatccatc actgggctct atgaaacccg tatcgacctt agtcaactgg ggggcgaccc 13681 tcccaagaag aagcgcaagg tctgagcgga cattcgattt atgccgttat gacttcctta 13741 aaaaagcctt tacgaatgaa agaaatggaa ttagacttgt tatgtagttg attctacaat 13801 ggattatgat tcctgaactt caaatccgct gttcattatt aatctcagct cttcccgtaa 13861 agccaatgtt gaaactattc gtaaatgtac ctcgttttgc gtgtaccttg cttatcacgt 13921 gatattacat gacctggaca gagttctgcg cgaaagtcat aacgtaaatc ccgggcggta 13981 ggtgcgtccc gggcggaagg tagttttctc gtccacccca acgcgtttat caacctcaac 14041 tttcaacaac catcatgcca ccaaaagcgc gtaaaacaaa gcgagatttg attgagcaag 14101 agggcaggat ccaatgcgcg attcaagaca ttaaaaatgg aaaatttcaa aaaattgcgc 14161 ccgcagcgcg tgcatacaaa attcatccca atacaagtgt aatgctagtg gaggtcaaca 14221 catcaatgcc tattttggtt tagtcgtcca ggcggtgagc acaaaatttg tgtcgtttga 14281 caagatggtt catttaggca actggtcaga tcagccccac ttgtagcagt agcggcggcg 14341 ctcgaagtgt gactcttatt agcagacagg aacgaggaca ttattatcat ctgctgcttg 14401 gtgcacgata acttggtgcg tttgtcaagc aaggtaagtg gacgacccgg tcataccttc 14461 ttaagttcgc ccttcctccc tttatttcag attcaatctg acttacctat tctacccaag 14521 catccaaatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg 14581 agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt 14641 cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt 14701 gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg 14761 gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg 14821 cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt 14881 cgtggccgag gagcaggact gaccgacgcc gaccaacacc gccggtccga cgcggcccga 14941 cgggtccgag gagcttgaga tccacttaac gttactgaaa tcatcaaaca gcttgacgaa 15001 tctggatata agatcgttgg tgtcgatgtc agctccggag ttgagacaaa tggtgttcag 15061 gatctcgata agatacgttc atttgtccaa gcagcaaaga gtgccttcta gtgatttaat 15121 agctccatgt caacaagaat aaaacgcgtt tcgggtttac ctcttccaga tacagctcat 15181 ctgcaatgca ttaatgcatt ggacctcgca accctagtac gcccttcagg ctccggcgaa 15241 gcagaagaat agcttagcag agtctatttt cattttcggg agacgagatc aagcagatca 15301 acggtcgtca agagacctac gagactgagg aatccgctct tggctccacg cgactatata 15361 tttgtctcta attgtacttt gacatgctcc tcttctttac tctgatagct tgactatgaa 15421 aattccgtca ccagcccctg ggttcgcaaa gataattgca ctgtttcttc cttgaactct 15481 caagcctaca ggacacacat tcatcgtagg tataaacctc gaaaatcatt cctactaaga 15541 tgggtataca atagtaacca tgcatggttg cctagtgaat gctccgtaac acccaatacg 15601 ccggccgaaa cttttttaca actctcctat gagtcgttta cccagaatgc acaggtacac 15661 ttgtttag //