质粒名称: | pLenti-HF1RA-P2A-GFP-PGK-Puro |
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出品公司: | 爱迪基因 |
目录编号: | 110866 |
存储实验室: | Lukas Dow |
载体骨架基本信息 |
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载体名称: | Adapted from lentiCRISPRv1 |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | 6296 bp |
完整质粒大小: | -- |
载体修饰: | -- |
载体类型: | 慢病毒表达 |
筛选标记: | 嘌呤霉素;GFP |
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-HF1RA,HF1RA |
物种来源: | Synthetic |
插入大小: | 4227 bp |
基因突变: | R661A, Q695A, Q926A, and NLS sequence at the N-terminus |
GenBank编号 | -- |
启动子 | EF1s |
标签/融合蛋白: | FLAG(插入N端子) |
克隆方法: | Gibson克隆 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | TAAGTGCAGTAGTCGCCGTG |
3' 测序引物 | TTAAGAATACCAGTCAATCTTTC |
文献引用方法 |
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材料和方法部分: | pLenti-HF1RA-P2A-GFP-PGK-Puro was a gift from Lukas Dow (Addgene plasmid # 110866 ; http://n2t.net/addgene:110866 ; RRID:Addgene_110866) |
参考文献部分: | Optimized base editors enable efficient editing in cells, organoids and mice. Zafra MP, Schatoff EM, Katti A, Foronda M, Breinig M, Schweitzer AY, Simon A, Han T, Goswami S, Montgomery E, Thibado J, Kastenhuber ER, Sanchez-Rivera FJ, Shi J, Vakoc CR, Lowe SW, Tschaharganeh DF, Dow LE. Nat Biotechnol. 2018 Jul 3. pii: nbt.4194. doi: 10.1038/nbt.4194. 10.1038/nbt.4194 PubMed 29969439 |
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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 pLenti-HF1RA-P2A-GFP-PGK-Puro 12448 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Lentiviral vector for constitutive expression of Cas9-HF1RA-P2A-GFP in mammalian cells (codon optimized). ACCESSION . VERSION . KEYWORDS pLenti-HF1RA-P2A-GFP-PGK-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12448) AUTHORS Zafra MP, Schatoff EM, Katti A, Foronda M, Breinig M, Schweitzer AY, Simon A, Han T, Goswami S, Montgomery E, Thibado J, Kastenhuber ER, Sanchez-Rivera FJ, Shi J, Vakoc CR, Lowe SW, Tschaharganeh DF, Dow LE TITLE Optimized base editors enable efficient editing in cells, organoids and mice. JOURNAL Nat Biotechnol. 2018 Jul 3. pii: nbt.4194. doi: 10.1038/nbt.4194. PUBMED 29969439 REFERENCE 2 (bases 1 to 12448) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..12448 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 222..402 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 449..574 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1067..1300 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." misc_feature 1713..1830 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 1899..2110 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 2130..2139 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2139..2162 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 2169..2189 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2214..6314 /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 KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6315..6362 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 6372..6428 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 6429..7148 /codon_start=1 /product="enhanced GFP" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(6474..6495) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(6735..6754) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 7082..7103 /label=EGFP-C /note="EGFP, forward primer" promoter 7160..7659 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(7195..7218) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 7592..7611 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 7680..8279 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(7680..7699) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 8176..8196 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 8295..8883 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(8348..8368) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(8766..8777) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 8955..9188 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 9266..9387 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(9303..9322) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 9357..9376 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 9427..9562 /label=SV40 ori /note="SV40 origin of replication" primer_bind 9489..9508 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(9582..9601) /label=T7 /note="T7 promoter, forward primer" promoter complement(9583..9601) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(9611..9628) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(9611..9627) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9620..9642) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 9769..10224 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9856..9875) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 10066..10087 /label=F1ori-F /note="F1 origin, forward primer" promoter 10250..10354 /gene="bla" /label=AmpR promoter CDS 10355..11215 /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(10573..10592) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 11386..11974 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 11875..11894 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 12128..12145 /label=L4440 /note="L4440 vector, forward primer" protein_bind 12262..12283 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 12298..12328 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12336..12352 /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)." primer_bind 12341..12363 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 12360..12376 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 12360..12376 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 12395..12415 /label=T3 /note="T3 promoter, forward primer" promoter 12397..12415 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 12443..221 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" ORIGIN 1 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac 61 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct 121 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt 181 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc 241 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 301 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 361 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga 421 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc 481 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg 541 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc 601 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat 661 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc 721 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga 781 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat 841 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac 901 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga 961 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca 1021 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc 1081 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac 1141 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg 1201 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa 1261 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg 1321 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg 1381 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa 1441 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat 1501 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt 1561 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg 1621 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc 1681 acctcccaac cccgagggga cccagaccac aattttaaaa gaaaaggggg gattgggggg 1741 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta 1801 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca 1861 ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac 1921 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg 1981 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg 2041 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc 2101 cagaacacag gtgtcgtgac gcgggatccg ccaccatgga ttacaaagac gatgacgata 2161 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga 2221 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg 2281 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca 2341 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc 2401 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag 2461 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt 2521 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg 2581 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg 2641 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt 2701 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc 2761 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca 2821 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa 2881 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc 2941 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac 3001 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg 3061 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg 3121 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga 3181 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc 3241 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg 3301 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg 3361 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga 3421 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc 3481 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc 3541 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct 3601 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca 3661 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca 3721 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc 3781 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc 3841 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc 3901 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg 3961 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca 4021 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga 4081 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg 4141 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc gccctgagcc 4201 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga 4261 agtccgacgg cttcgccaac agaaacttca tggccctgat ccacgacgac agcctgacct 4321 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca 4381 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg 4441 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg 4501 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga 4561 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca 4621 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg 4681 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc 4741 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc 4801 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc 4861 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg 4921 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa 4981 cccgggccat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg 5041 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt 5101 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg 5161 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc 5221 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca 5281 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga 5341 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg 5401 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc 5461 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg 5521 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga 5581 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc 5641 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc 5701 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag 5761 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt 5821 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa 5881 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga 5941 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc 6001 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg 6061 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag 6121 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct 6181 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg 6241 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc 6301 agctgggagg cgacaagcgt cctgctgcta ctaagaaagc tggtcaagct aagaaaaaga 6361 aaggcagcgg cgccaccaac ttcagcctgc tgaagcaggc cggcgacgtg gaggagaacc 6421 ccggccccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg 6481 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc tggcgagggc gagggcgatg 6541 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct 6601 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc 6661 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca 6721 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg 6781 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc 6841 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc 6901 agaagaacgg catcaaggcg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc 6961 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg 7021 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc 7081 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt 7141 acaagtaagc tagcctaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgc 7201 gctttagcag ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat 7261 tccacatcca ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct 7321 tctactcctc ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg 7381 tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa 7441 tggaagcggg taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg 7501 gctcagaggc tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg 7561 gcgggcgccc gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct 7621 gccgcgctgt tctcctcttc ctcatctccg ggcctttcga cctgcagccc aagcttacca 7681 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac 7741 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc 7801 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca 7861 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga 7921 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt 7981 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc 8041 ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca 8101 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg 8161 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg 8221 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgat 8281 tctagaaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc 8341 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg 8401 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc 8461 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg 8521 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg 8581 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga 8641 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct 8701 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg 8761 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc 8821 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc 8881 cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa 8941 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc 9001 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg 9061 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg 9121 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat 9181 ctctagcagt acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa 9241 gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata 9301 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 9361 tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact 9421 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta 9481 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag 9541 tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt 9601 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 9661 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 9721 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta 9781 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 9841 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 9901 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 9961 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 10021 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 10081 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 10141 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 10201 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc 10261 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 10321 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 10381 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 10441 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 10501 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 10561 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 10621 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 10681 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 10741 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 10801 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 10861 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 10921 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 10981 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 11041 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 11101 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 11161 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 11221 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 11281 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 11341 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 11401 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 11461 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 11521 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 11581 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 11641 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 11701 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 11761 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 11821 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 11881 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 11941 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 12001 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 12061 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 12121 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 12181 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 12241 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 12301 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 12361 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca 12421 aaagctggag ctgcaagctt aatgtagt //