质粒名称: | pCAG-SpCas9-2A-GFP-noITR |
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出品公司: | Addgene |
目录编号: | 118415 |
存储实验室: | Mark Denham |
载体骨架基本信息 |
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载体名称: | pSpCas9(BB)-2A-GFP (PX458) |
载体出品公司: | -- |
空载体大小: | -- |
完整质粒大小: | 10125 bp |
载体修饰: | 通过用NotI和SbfI限制性消化去除质粒ITR区域以去除145bp的片段。在doi:10.3389/fcell.2018.00054中克隆了一个含有BamHI位点的片段,该位点具有与NotI和SbfI相容的两个粘性末端 |
载体类型: | 哺乳动物表达,CRISPR |
筛选标记: | -- |
载体抗性: | 氨苄青霉素,100μg/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: | Cas9 |
物种来源: | -- |
插入大小: | -- |
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: | pCAG-SpCas9-2A-GFP-noITR was a gift from Mark Denham (Addgene plasmid # 118415 ; http://n2t.net/addgene:118415 ; RRID:Addgene_118415) |
参考文献部分: | A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity. Hojland Knudsen C, Asgrimsdottir ES, Rahimi K, Gill KP, Frandsen S, Hvolbol Buchholdt S, Chen M, Kjems J, Febbraro F, Denham M. Front Cell Dev Biol. 2018 May 15;6:54. doi: 10.3389/fcell.2018.00054. eCollection 2018. 10.3389/fcell.2018.00054 PubMed 29868584 |
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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 pCAG-SpCas9-2A-GFP-noITR 10125 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Cas9 GFP plasmid constitutively expressed from CAG promoter. AAV repeat region removed to increase expression. ACCESSION . VERSION . KEYWORDS pCAG-SpCas9-2A-GFP-noITR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10125) AUTHORS Hojland Knudsen C, Asgrimsdottir ES, Rahimi K, Gill KP, Frandsen S, Hvolbol Buchholdt S, Chen M, Kjems J, Febbraro F, Denham M TITLE A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity. JOURNAL Front Cell Dev Biol. 2018 May 15;6:54. doi: 10.3389/fcell.2018.00054. eCollection 2018. PUBMED 29868584 REFERENCE 2 (bases 1 to 10125) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..10125 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 6..246 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 6..26 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 177..196 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 273..348 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 465..844 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 846..1121 /label=chicken beta-actin promoter intron 1122..2138 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 2062..2084 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 2146..2165 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" regulatory 2205..2214 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2214..2279 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 2286..2306 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2331..6431 /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6432..6479 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 6495..6548 /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 6549..7262 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 6549..7262 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(6591..6612) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(6852..6871) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 7199..7220 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(7290..7307) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 7296..7503 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 7608..8063 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7695..7714) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7905..7926 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(8080..8099) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8199..8221 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(8259..8277) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 8345..8449 /gene="bla" /label=AmpR promoter CDS 8450..9310 /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 complement(8668..8687) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 9481..10069 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9970..9989 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta 61 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac 121 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact 181 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga 241 aaggacgaaa caccgggtct tcgagaagac ctgttttaga gctagaaata gcaagttaaa 301 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgtttta 361 gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc gccaattctg 421 cagacaaatg gctctagagg tacccgggga tctatatata tgtcgacatt gattattgac 481 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 541 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 601 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 661 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 721 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 781 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 841 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc 901 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg 961 gggggggcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag 1021 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc 1081 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc 1141 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg 1201 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct 1261 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg 1321 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga 1381 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc 1441 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg 1501 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag 1561 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg 1621 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc 1681 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg 1741 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga 1801 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc 1861 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg 1921 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc 1981 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc 2041 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct 2101 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta 2161 ttgtgctgtc tcatcatttt ggcaaagaat tcctcgagac cggtgccacc atggactata 2221 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 2281 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 2341 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 2401 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 2461 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 2521 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 2581 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 2641 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 2701 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 2761 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 2821 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 2881 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 2941 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 3001 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 3061 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 3121 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 3181 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 3241 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 3301 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 3361 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 3421 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 3481 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 3541 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 3601 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 3661 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 3721 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 3781 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 3841 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 3901 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 3961 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 4021 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 4081 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 4141 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 4201 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 4261 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 4321 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 4381 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 4441 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 4501 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 4561 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 4621 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 4681 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 4741 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 4801 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 4861 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 4921 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 4981 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 5041 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 5101 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 5161 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 5221 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 5281 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 5341 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 5401 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 5461 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 5521 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 5581 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 5641 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 5701 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 5761 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 5821 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 5881 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 5941 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 6001 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 6061 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 6121 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 6181 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 6241 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 6301 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 6361 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 6421 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg 6481 aattcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc 6541 ctggcccagt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc 6601 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca 6661 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc 6721 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca 6781 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca 6841 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca 6901 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg 6961 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga 7021 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc 7081 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca 7141 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca 7201 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca 7261 aggaattcta actagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 7321 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 7381 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 7441 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg 7501 ggagcggccg cttttggatc ctttacctgc aggggcgcct gatgcggtat tttctcctta 7561 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag 7621 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 7681 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 7741 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 7801 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata 7861 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 7921 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc 7981 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 8041 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc 8101 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 8161 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 8221 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 8281 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 8341 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 8401 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 8461 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 8521 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 8581 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 8641 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 8701 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 8761 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 8821 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 8881 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 8941 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 9001 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 9061 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 9121 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat 9181 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 9241 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 9301 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 9361 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 9421 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 9481 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 9541 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 9601 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 9661 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 9721 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 9781 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 9841 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 9901 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 9961 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 10021 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 10081 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctca //
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