质粒名称: | pPB-C6F/TdTomato |
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出品公司: | Addgene |
目录编号: | 140826 |
存储实验室: | Hideyuki Okano |
载体骨架基本信息 |
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载体名称: | pTRE-Tight-BI |
载体出品公司: | Clontech |
空载体大小: | -- |
完整质粒大小: | 16425 bp |
载体修饰: | -- |
载体类型: | 哺乳动物表达 |
筛选标记: | 嘌呤霉素 |
载体抗性: | 氨苄青霉素,100μg/mL |
生长温度: | 37 ℃ |
宿主菌: | NEB Stable |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称1: | OCT4 |
物种来源1: | H. sapiens (human) |
插入大小1: | 1080 bp |
GenBank编号1 | NM_002701.6 |
插入基因名称2: | SOX2 |
物种来源2: | H. sapiens (human) |
插入大小2: | 954 bp |
GenBank编号2 | NM_003106.4 |
插入基因名称3: | KLF4 |
物种来源3: | H. sapiens (human) |
插入大小3: | 1410 bp |
GenBank编号3 | NM_004235.6 |
插入基因名称4: | C-MYC |
物种来源4: | H. sapiens (human) |
插入大小4: | 1320 bp |
GenBank编号4 | NM_002467.6 |
文献引用方法 |
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材料和方法部分: | pPB-C6F/TdTomato was a gift from Hideyuki Okano (Addgene plasmid # 140826 ; http://n2t.net/addgene:140826 ; RRID:Addgene_140826) |
参考文献部分: | Naive-like ESRRB(+) iPSCs with the Capacity for Rapid Neural Differentiation. Kisa F, Shiozawa S, Oda K, Yoshimatsu S, Nakamura M, Koya I, Kawai K, Suzuki S, Okano H. Stem Cell Reports. 2017 Dec 12;9(6):1825-1838. doi: 10.1016/j.stemcr.2017.10.008. Epub 2017 Nov 9. 10.1016/j.stemcr.2017.10.008 PubMed 29129686 |
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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 pPB-C6F/TdTomato 16441 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION OCT4, SOX2, KLF4, C-MYC, KLF2, NANOG (C6F) and tdTomato under the control of TRE (Tet-ON). ACCESSION . VERSION . KEYWORDS pPB-C6F/TdTomato SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 16441) AUTHORS Kisa F, Shiozawa S, Oda K, Yoshimatsu S, Nakamura M, Koya I, Kawai K, Suzuki S, Okano H TITLE Naive-like ESRRB(+) iPSCs with the Capacity for Rapid Neural Differentiation. JOURNAL Stem Cell Reports. 2017 Dec 12;9(6):1825-1838. doi: 10.1016/j.stemcr.2017.10.008. Epub 2017 Nov 9. PUBMED 29129686 REFERENCE 2 (bases 1 to 16441) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..16441 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(3..458) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(140..161) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 352..371 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 600..616 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 623..641 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" repeat_region 660..694 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind 1097..1113 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 1120..1138 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" polyA_signal 1238..1359 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind 1303..1322 /label=SV40pA-R /note="SV40 polyA, reverse primer" CDS complement(1496..2926) /codon_start=1 /product="tandem dimeric (pseudo-monomeric) derivative of DsRed (Shaner et al., 2004)" /label=tdTomato /note="mammalian codon-optimized" /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE RSEGRHHLFLYGMDELYK" regulatory 2923..2932 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" misc_feature 2946..3498 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" misc_feature 2948..3521 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" CDS complement(4546..4611) /codon_start=1 /product="2A peptide from foot-and-mouth disease virus polyprotein" /label=F2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="VKQTLNFDLLKLAGDVESNPGP" regulatory 5697..5706 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter 5760..6147 /label=bidirectional TRE promoter /note="Tet-responsive bidirectional promoter PTight-BI, consisting of seven tet operator sequences flanked on each side by the minimal CMV promoter" protein_bind 5834..5852 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5870..5888 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5905..5923 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5941..5959 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5977..5995 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6012..6030 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 6048..6066 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 6164..6184 /label=T3 /note="T3 promoter, forward primer" promoter 6166..6184 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" CDS 7314..7379 /codon_start=1 /product="2A peptide from foot-and-mouth disease virus polyprotein" /label=F2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="VKQTLNFDLLKLAGDVESNPGP" CDS 8826..8891 /codon_start=1 /product="2A peptide from foot-and-mouth disease virus polyprotein" /label=F2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="VKQTLNFDLLKLAGDVESNPGP" misc_feature 9884..10457 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" CDS 10502..11821 /codon_start=1 /product="human c-Myc proto-oncogene" /label=c-Myc /translation="MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAP SEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTE LLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPN PARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDS LLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGS PSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNR KCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKA TAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA" misc_feature 11848..12434 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" CDS 12435..13034 /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(12435..12454) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 12931..12951 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 13176..13409 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(13549..13566) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 13555..13779 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(13826..13842) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 13850..13866 /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(13874..13904) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 13919..13940 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." repeat_region 14167..14229 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" promoter complement(14253..14271) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(14292..14308) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 14316..14332 /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(14340..14370) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 14385..14406 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(14523..14540) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(14694..15282) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(14774..14793) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(15453..16313) /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 16076..16095 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(16314..16418) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccttaac 661 cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt 721 ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc 781 cgtgaggcgt gcttgtcaat gaggtaagtg tcactgattt tgaactataa cgaccgcgtg 841 agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat 901 tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta 961 tagatgtata ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 1021 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt 1081 tcccagtcac gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc 1141 gaattgggcc ctctagatgc atgctcgacg gtatacagac atgataagat acattgatga 1201 gtttggacaa accacaacta gaatgcactc gacgcgttaa gatacattga tgagtttgga 1261 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt 1321 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat 1381 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac 1441 aaatgtggta tggctgatta tgatcagtta tctagatgca tgctcgagcg gccgcctact 1501 tgtacagctc gtccatgccg tacaggaaca ggtggtggcg gccctcggag cgctcgtact 1561 gttccacgat ggtgtagtcc tcgttgtggg aggtgatgtc cagcttggtg tccacgtagt 1621 agtagccggg cagttgcacg ggcttcttgg ccatgtagat ggtcttgaac tccaccaggt 1681 agtggccgcc gtccttcagc ttcagggcct ggtggatctc gcccttcagc acgccgtcgc 1741 gggggtacag gcgctcggtg gaggcctccc agcccatggt cttcttctgc attacggggc 1801 cgtcgggggg gaagttggtg ccgcgcatct tcaccttgta gatcagcgtg ccgtcctgca 1861 gggaggagtc ctgggtcacg gtcaccagac cgccgtcctc gaagttcatc acgcgctccc 1921 acttgaagcc ctcggggaag gacagcttct tgtaatcggg gatgtcggcg gggtgcttca 1981 cgtacgcctt ggagccgtac atgaactggg gggacaggat gtcccaggcg aagggcaggg 2041 ggccgccctt ggtcaccttc agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc 2101 cctcgccctc gatctcgaac tcgtggccgt tcatggagcc ctccatgcgc accttgaagc 2161 gcatgaactc tttgatgacg gccatgttgt tgtcctcgga ggaggcggtg ccggagctgc 2221 cgctgccggt gctgccggtg ccatgcccca ggaacaggtg gtggcggccc tcggagcgct 2281 cgtactgttc cacgatggtg tagtcctcgt tgtgggaggt gatgtccagc ttggtgtcca 2341 cgtagtagta gccgggcagt tgcacgggct tcttggccat gtagatggtc ttgaactcca 2401 ccaggtagtg gccgccgtcc ttcagcttca gggcctggtg gatctcgccc ttcagcacgc 2461 cgtcgcgggg gtacaggcgc tcggtggagg cctcccagcc catggtcttc ttctgcatta 2521 cggggccgtc gggggggaag ttggtgccgc gcatcttcac cttgtagatc agcgtgccgt 2581 cctgcaggga ggagtcctgg gtcacggtca ccagaccgcc gtcctcgaag ttcatcacgc 2641 gctcccactt gaagccctcg gggaaggaca gcttcttgta atcggggatg tcggcggggt 2701 gcttcacgta cgccttggag ccgtacatga actgggggga caggatgtcc caggcgaagg 2761 gcagggggcc gcccttggtc accttcagct tggcggtctg ggtgccctcg taggggcggc 2821 cctcgccctc gccctcgatc tcgaactcgt ggccgttcat ggagccctcc atgcgcacct 2881 tgaagcgcat gaactctttg atgacctcct cgcccttgct caccatggtg gcggatccgt 2941 ggccatatta tcatcgtgtt tttcaaagga aaaccacgtc cccgtggttc ggggggccta 3001 gacgtttttt taacctcgac taaacacatg taaagcatgt gtaccgaggc cccagatcag 3061 atcccataca atggggtacc ttctgggcat ccttcagccc cttgttgaat acgcttgagg 3121 agagccattt gactctttcc acaactatcc aactcacaac gtggcactgg ggttgtgccg 3181 cctttgcagg tgtatcttat acacgtggct tttggccgca gaggcacctg tcgccaggtg 3241 gggggttccg ctgcctgcaa agggtcgcta cagacgttgt ttgtcttcaa gaagcttcca 3301 gaggaactgc ttccttcacg acattcaaca gaccttgcat tcctttggcg agaggggaaa 3361 gacccctagg aatgctcgtc aagaagacag ggccaggttt ccgggccctc acattgccaa 3421 aagacggcaa tatggtggaa aataacatat agacaaacgc acaccggcct tattccaagc 3481 ggcttcggcc agtaacgtta gggggggggg agggagaggg gcggactgtg gaaaaaaaag 3541 ggagatccgc tagaagggcg aattcgaatc cggagacgta cgaccggtta acaggcctta 3601 agcgctagat gcatgctcga ctcacacgtc ttcaggttgc atgttcatgg agtagtttag 3661 gaataaatcc atggtttgtg gagtactaaa atacctagtg gtctgctgta ttacattaag 3721 gccttcccca gcagcttcca aagcagcctc caagtcactg gcaggagaat ttggctggaa 3781 ctgcatgcag gactgcagag attcctctcc acagttatag aagggactgt tccaggcctg 3841 attgttccag gattgggtgc accaggtctg agtgttccag gagtggttgc tccaggactg 3901 gatgttctgg gtctggttgc tccaggttga attgttccag gtctggttgc tccacattgg 3961 aaggttccca gtcgggttca ccaggcatcc ctggtggtag gaagagtaga ggctggggta 4021 ggtaggtgct gaggccttct gcgtcacacc attgctattc ttcggccagt tgtttttctg 4081 ccacctctta gatttcattc tctggttctg gaaccaggtc ttcacctgtt tgtagctgag 4141 gttcaggatg ttggagagtt cttgcatctg ctggaggctg aggtatttct gtctctgaaa 4201 tctatcattg agtacacaca gctgggtgga agagaacaca gttctggtct tctgtttctt 4261 gactgggacc ttgtcttcct tttttgcgac actattctct gcagaagtgg gttgtttgcc 4321 tttgggactg gtggaagaat cagggctgtc ctgaataagc agatccatgg aggagggaag 4381 aggagagaca gtctccgtgt gaggcatctc agcagaagac atttgcaagg atggatagtt 4441 ttcttcaggc ccacaaatca caggcatagg tgaagattct ttacagtcgg atgcttcaaa 4501 gcaaggcaag ctttggggac aagctggatc cacactcatg atatccggcc ctgggttgga 4561 ctccacgtct cccgccaact tgagaaggtc aaaattcaaa gtctgtttca ccggtgcccg 4621 cttagcgcga ggatccatgt gccgtttcat gtgcagcgcc aggtgatcgg agcgcgagaa 4681 ggcacgatcg cacagatggc actggaatgg ccggtggccc gtgtgctttc ggtagtggcg 4741 cgtgagctcg tctgagcgcg caaacttcca gccgcagccg tcccagttgc agtggtaggg 4801 cttctcacct gtgtgcgtgc gcagatgcgc cttcagatgc gaactcttgg tgtaggtctt 4861 gccgcagccc gcgtagctgc aggtgtgagt ggcggtgcgt ttgcggggcc aagagcggcg 4921 gccgcgcttt ggcttggcct ccagcagctc cagcggggac gcaggcggcg tgaggagacc 4981 gcgggcggcg gggggcgcca ggcccagggc tgccgcggcg gcggccgcgt cgtcgaagag 5041 accgaaggct gggggcgcag gcggcgcgta atgcaggccg ggcccgggcg cgccgaaacc 5101 agggccaccg aaaggcggcg ggaaggaggc gcgcggaccg ggcgcgggca ggcgcgcggg 5161 gccgtcgggg ctgagcggcg gtgtgtcggg cggcggcggg gggcggcccc cgggacctcg 5221 catgcacgaa gccgccgggc ccggggcgcc ctcgcgcttg aggccgcgcg gtccacgggt 5281 cagcccgggg gcgcagccgt agccgccgcc gccgtccgct tcagggggct cggccttgac 5341 caggcggccg ggcggcgcca gcagaaagcg gccgtgcagt gcgggccccg gcggcgcatc 5401 cagctcgggt cgcagcagct cagacaccag gccacccgcg ggggcgctgt agggcggggg 5461 cgcgccgggt tcggggtaat agaacgcagg cggcgggggc ggcggcggcg gctccggggc 5521 ggcctcggcg cccaggccat ccagccccat ggacaggatg aagtccagca cgctgttgag 5581 gtcgtcgtcg gtgccgccgg actcgggttc ggcgcgcggc cagcgctcct gcaggccgcg 5641 ctcgcggcac gggctggcga aagtggagaa ggacggcagg atgggttcac tcagcgccat 5701 ggtggctagc acgcgtcagc tgactagagg atccccgggt accgagctcg aaatctccag 5761 gcgatctgac ggttcactaa acgagctctg cttatatagg cctcccaccg tacacgccta 5821 cctcgacata cgttctctat cactgatagg gagtaaactc gacatacgtt ctctatcact 5881 gatagggata aactcgacat acgttctcta tcactgatag ggagtaaact cgacatacgt 5941 tctctatcac tgatagggag taaactcgac atacgttctc tatcactgat agggagtaaa 6001 ctcgacatcg ttctctatca ctgataggga gtaaactcga catacgttct ctatcactga 6061 tagggagtaa actcgagtat gtcgaggtgg cgtgtacggt gggaggccta tataagcaga 6121 gctcgtttag tgaaccgtca gatcgcctgg agaattcgcc taggcaatta accctcacta 6181 aagggaaggc cgcctgggcc acgcgtgcca tggcgggaca cctggcttcg gatttcgcct 6241 tctcgccccc tccaggtggt ggaggtgatg ggccaggggg gccggagccg ggctgggttg 6301 atcctcggac ctggctaagc ttccaaggcc ctcctggagg gccaggaatc gggccggggg 6361 ttgggccagg ctctgaggtg tgggggattc ccccatgccc cccgccgtat gagttctgtg 6421 gggggatggc gtactgtggg ccccaggttg gagtggggct agtgccccaa ggcggcttgg 6481 agacctctca gcctgagggt gaagcaggag tcggggtgga gagcaactcc gatggggcct 6541 ccccggagcc ctgcaccgtc acccctggtg ccgtgaagct ggagaaggag aagctggagc 6601 aaaacccgga ggagtcccag gacatcaaag ctctgcagaa agaactcgag caatttgcca 6661 agctcctgaa gcagaagagg atcaccctgg gatatacaca ggccgatgtg gggctcaccc 6721 tgggggttct atttgggaag gtattcagcc aaacgaccat ctgccgcttt gaggctctgc 6781 agcttagctt caagaacatg tgtaagctgc ggcccttgct gcagaagtgg gtggaggaag 6841 ctgacaacaa tgaaaatctt caggagatat gcaaagcaga aaccctcgtg caggcccgaa 6901 agagaaagcg aaccagtatc gagaaccgag tgagaggcaa cctggagaat ttgttcctgc 6961 agtgcccgaa acccacactg cagcagatca gccacatcgc ccagcagctt gggctcgaga 7021 aggatgtggt ccgagtgtgg ttctgtaacc ggcgccagaa gggcaagcga tcaagcagcg 7081 actatgcaca acgagaggat tttgaggctg ctgggtctcc tttctcaggg ggaccagtgt 7141 cctttcctct ggccccaggg ccccattttg gtaccccagg ctatgggagc cctcacttca 7201 ctgcactgta ctcctcggtc cctttccctg agggggaagc ctttccccct gtctctgtca 7261 ccactctggg ctctcccatg cattcaaacg gggcgcgtaa aattgtcgct cctgtcaaac 7321 aaactcttaa ctttgattta ctcaaactgg ctggggatgt agaaagcaat ccaggtccag 7381 gggcgcgtgg tatggctgtc agcgacgcgc tgctcccatc tttctccacg ttcgcgtctg 7441 gcccggcggg aagggagaag acactgcgtc aagcaggtgc cccgaataac cgctggcggg 7501 aggagctctc ccacatgaag cgacttcccc cagtgcttcc cggccgcccc tatgacctgg 7561 cggcggcgac cgtggccaca gacctggaga gcggcggagc cggtgcggct tgcggcggta 7621 gcaacctggc gcccctacct cggagagaga ccgaggagtt caacgatctc ctggacctgg 7681 actttattct ctccaattcg ctgacccatc ctccggagtc agtggccgcc accgtgtcct 7741 cgtcagcgtc agcctcctct tcgtcgtcgc cgtcgagcag cggccctgcc agcgcgccct 7801 ccacctgcag cttcacctat ccgatccggg ccgggaacga cccgggcgtg gcgccgggcg 7861 gcacgggcgg aggcctcctc tatggcaggg agtccgctcc ccctccgacg gctcccttca 7921 acctggcgga catcaacgac gtgagcccct cgggcggctt cgtggccgag ctcctgcggc 7981 cagaattgga cccggtgtac attccgccgc agcagccgca gccgccaggt ggcgggctga 8041 tgggcaagtt cgtgctgaag gcgtcgctga gcgcccctgg cagcgagtac ggcagcccgt 8101 cggtcatcag cgtcagcaaa ggcagccctg acggcagcca cccggtggtg gtggcgccct 8161 acaacggcgg gccgccgcgc acgtgcccca agatcaagca ggaggcggtc tcttcgtgca 8221 cccacttggg cgctggaccc cctctcagca atggccaccg gccggctgca cacgacttcc 8281 ccctggggcg gcagctcccc agcaggacta ccccgaccct gggtcttgag gaagtgctga 8341 gcagcaggga ctgtcaccct gccctgccgc ttcctcccgg cttccatccc cacccggggc 8401 ccaattaccc atccttcctg cccgatcaga tgcagccgca agtcccgccg ctccattacc 8461 aagagctcat gccacccggt tcctgcatgc cagaggagcc caagccaaag aggggaagac 8521 gatcgtggcc ccggaaaagg accgccaccc acacttgtga ttacgcgggc tgcggcaaaa 8581 cctacacaaa gagttcccat ctcaaggcac acctgcgaac ccacacaggt gagaaacctt 8641 accactgtga ctgggacggc tgtggatgga aattcgcccg ctcagatgaa ctgaccaggc 8701 actaccgtaa acacacgggg caccgcccgt tccagtgcca aaaatgcgac cgagcatttt 8761 ccaggtcgga ccacctcgcc ttacacatga agaggcattt tggggcgcgt aaaattgtcg 8821 ctcctgtcaa acaaactctt aactttgatt tactcaaact ggctggggat gtagaaagca 8881 atccaggtcc aggggcgcgt ggtatgtaca acatgatgga gacggagctg aagccgccgg 8941 gcccgcagca aacttcgggg ggcggcggcg gcaactccac cgcggcggcg gccggcggca 9001 accagaaaaa cagcccggac cgcgtcaagc ggcccatgaa tgccttcatg gtgtggtccc 9061 gcgggcagcg gcgcaagatg gcccaggaga accccaagat gcacaactcg gagatcagca 9121 agcgcctggg cgccgagtgg aaacttttgt cggagacgga gaagcggccg ttcatcgacg 9181 aggctaagcg gctgcgagcg ctgcacatga aggagcaccc ggattataaa taccggcccc 9241 ggcggaaaac caagacgctc atgaagaagg ataagtacac gctgcccggc gggctgctgg 9301 cccccggcgg caatagcatg gcgagcgggg tcggggtggg cgccggcctg ggcgcgggcg 9361 tgaaccagcg catggacagt tacgcgcaca tgaacggctg gagcaacggc agctacagca 9421 tgatgcagga ccagctgggc tacccgcagc acccgggcct caatgcgcac ggcgcagcgc 9481 agatgcagcc catgcaccgc tacgacgtga gcgccctgca gtacaactcc atgaccagct 9541 cgcagaccta catgaacggc tcgcccacct acagcatgtc ctactcgcag cagggcaccc 9601 ctggcatggc tcttggctcc atgggttcgg tggtcaagtc cgaggccagc tccagccccc 9661 ctgtggttac ctcttcctcc cactccaggg cgccctgcca ggccggggac ctccgggaca 9721 tgatcagcat gtatctcccc ggcgccgagg tgccggaacc cgccgccccc agcagacttc 9781 acatgtccca gcactaccag agcggcccgg tgcccggcac ggccattaac ggcacactgc 9841 ccctctcaca catgtgagtt taaatctagg gcggccaatt ccgcccctct cccccccccc 9901 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt 9961 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct 10021 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga 10081 atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga 10141 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac 10201 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag 10261 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc 10321 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg 10381 tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt 10441 gaaaaacacg atgataagct tgccacaacc cacaaggaga cgaccttcca tggcgcgtgg 10501 tatgcccctc aacgttagct tcaccaacag gaactatgac ctcgactacg actcggtgca 10561 gccgtatttc tactgcgacg aggaggagaa cttctaccag cagcagcagc agagcgagct 10621 gcagcccccg gcgcccagcg aggatatctg gaagaaattc gagctgctgc ccaccccgcc 10681 cctgtcccct agccgccgct ccgggctctg ctcgccctcc tacgttgcgg tcacaccctt 10741 ctcccttcgg ggagacaacg acggcggtgg cgggagcttc tccacggccg accagctgga 10801 gatggtgacc gagctgctgg gaggagacat ggtgaaccag agtttcatct gcgacccgga 10861 cgacgagacc ttcatcaaaa acatcatcat ccaggactgt atgtggagcg gcttctcggc 10921 cgccgccaag ctcgtctcag agaagctggc ctcctaccag gctgcgcgca aagacagcgg 10981 cagcccgaac cccgcccgcg gccacagcgt ctgctccacc tccagcttgt acctgcagga 11041 tctgagcgcc gccgcctcag agtgcatcga cccctcggtg gtcttcccct accctctcaa 11101 cgacagcagc tcgcccaagt cctgcgcctc gcaagactcc agcgccttct ctccgtcctc 11161 ggattctctg ctctcctcga cggagtcctc cccgcagggc agccccgagc ccctggtgct 11221 ccatgaggag acaccgccca ccaccagcag cgactctgag gaggaacaag aagatgagga 11281 agaaatcgat gttgtttctg tggaaaagag gcaggctcct ggcaaaaggt cagagtctgg 11341 atcaccttct gctggaggcc acagcaaacc tcctcacagc ccactggtcc tcaagaggtg 11401 ccacgtctcc acacatcagc acaactacgc agcgcctccc tccactcgga aggactatcc 11461 tgctgccaag agggtcaagt tggacagtgt cagagtcctg agacagatca gcaacaaccg 11521 aaaatgcacc agccccaggt cctcggacac cgaggagaat gtcaagaggc gaacacacaa 11581 cgtcttggag cgccagagga ggaacgagct aaaacggagc ttttttgccc tgcgtgacca 11641 gatcccggag ttggaaaaca atgaaaaggc ccccaaggta gttatcctta aaaaagccac 11701 agcatacatc ctgtccgtcc aagcagagga gcaaaagctc atttctgaag aggacttgtt 11761 gcggaaacga cgagaacagt tgaaacacaa acttgaacag ctacggaact cttgtgcgta 11821 agtttatctg cagaattccg cccccccccc ctctccctcc ccccccccta acgttactgg 11881 ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt 11941 gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc 12001 taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc 12061 agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg 12121 gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc 12181 tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa 12241 atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg 12301 tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa 12361 aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata 12421 atatggccac aaccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg 12481 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca 12541 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc 12601 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct 12661 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg 12721 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc 12781 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg 12841 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg 12901 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct 12961 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca 13021 agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac 13081 cccatgcatc gatttcgaac ccggggtacc tttaagacca atgacttaca aggcagctgt 13141 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaaag 13201 aagacaagat ctgctttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg 13261 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt 13321 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc 13381 cttttagtca gtgtggaaaa tctctagcag aattccagca cactggcggc cgttactagt 13441 ggatcatcat aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca 13501 tgatctatag atcctaagct tatcgatgat ctagagctcg ctgatcagcc tcgactgtgc 13561 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 13621 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 13681 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 13741 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacct 13801 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 13861 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 13921 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 13981 tgtcgtgcca gcggatcctt tgttacttta tagaagaaat tttgagtttt tgtttttttt 14041 taataaataa ataaacataa ataaattgtt tgttgaattt attattagta tgtaagtgta 14101 aatataataa aacttaatat ctattcaaat taataaataa acctcgatat acagaccgat 14161 aaaacacatg cgtcaatttt acgcatgatt atctttaacg tacgtcacaa tatgattatc 14221 tttctagggt taaagagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg 14281 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 14341 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 14401 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 14461 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 14521 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 14581 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 14641 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 14701 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 14761 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 14821 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 14881 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 14941 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 15001 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 15061 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 15121 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 15181 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 15241 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 15301 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 15361 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 15421 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 15481 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 15541 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 15601 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 15661 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 15721 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 15781 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 15841 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 15901 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 15961 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 16021 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 16081 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 16141 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 16201 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacgggaagg 16261 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 16321 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 16381 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 16441 c //