质粒名称: | pEBTet_NADP-Snifit-mito |
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出品公司: | 爱迪基因 |
目录编号: | 113919 |
存储实验室: | Kai Johnsson |
载体骨架基本信息 |
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载体名称: | pEBTet |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | 11448 bp |
完整质粒大小: | 14257 bp |
载体修饰: | -- |
载体类型: | 哺乳细胞表达 |
筛选标记: | 嘌呤霉素 |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | NADP-Snifit,hSPR-HaloTag7-Pro30-SNAPtag |
物种来源: | Synthetic |
插入大小: | 2349 bp |
基因突变: | -- |
GenBank编号 | -- |
启动子 | CMV TetO2 |
标签/融合蛋白: | -- |
克隆方法: | Gibson克隆 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | CMV_Fwd |
3' 测序引物 | BGH_Rev |
文献引用方法 |
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材料和方法部分: | pEBTet_NADP-Snifit-mito was a gift from Kai Johnsson (Addgene plasmid # 113919 ; http://n2t.net/addgene:113919 ; RRID:Addgene_113919) |
参考文献部分: | Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides. Sallin O, Reymond L, Gondrand C, Raith F, Koch B, Johnsson K. Elife. 2018 May 29;7. pii: 32638. doi: 10.7554/eLife.32638. 32638 [pii] PubMed 29809136 |
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载体基本信息
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载体简介
载体序列
LOCUS pEBTet_NADP-Snifit-mito 14157 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid encoding the mitochondrial localization of the FRET semi-synthetic biosensor measuring the ratio NADPH/NADP+ in mammalian cells. ACCESSION . VERSION . KEYWORDS pEBTet_NADP-Snifit-mito SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14157) AUTHORS Sallin O, Reymond L, Gondrand C, Raith F, Koch B, Johnsson K TITLE Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides. JOURNAL Elife. 2018 May 29;7. pii: 32638. doi: 10.7554/eLife.32638. PUBMED 29809136 REFERENCE 2 (bases 1 to 14157) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14157 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 12..86 /codon_start=1 /product="mitochondrial presequence of human cytochrome c oxidase subunit VIII (Rizutto et al., 1989)" /label=COX8 presequence /translation="MSVLTPLLLRGLTGSARRLPVPRAK" CDS 99..170 /codon_start=1 /product="mitochondrial presequence of human cytochrome c oxidase subunit VIII (Rizutto et al., 1989)" /label=COX8 presequence /translation="SVLTPLLLRGLTGSARRLPVPRAK" CDS 189..260 /codon_start=1 /product="mitochondrial presequence of human cytochrome c oxidase subunit VIII (Rizutto et al., 1989)" /label=COX8 presequence /translation="SVLTPLLLRGLTGSARRLPVPRAK" CDS 273..344 /codon_start=1 /product="mitochondrial presequence of human cytochrome c oxidase subunit VIII (Rizutto et al., 1989)" /label=COX8 presequence /translation="SVLTPLLLRGLTGSARRLPVPRAK" CDS 1167..2051 /codon_start=1 /product="modified bacterial dehalogenase that forms covalent bonds with chloroalkane derivatives" /label=HaloTag(R) /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG SEIARWLSTLEISG" primer_bind complement(2782..2799) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2788..2899 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3440..5229 /label=oriP /note="Epstein-Barr virus oriP replication origin (Yates et al., 2000)" primer_bind complement(7893..7911) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7979..8083 /gene="bla" /label=AmpR promoter CDS 8084..8944 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(8302..8321) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 9115..9703 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9604..9623 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind complement(9892..9912) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 9910..10207 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind 10139..10158 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 10284..10883 /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(10284..10303) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 10780..10800 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 10974..11108 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(11246..11265) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 11542..11803 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" intron 11920..12492 /label=-beta-globin intron /note="intron from rabbit beta-globin gene" primer_bind 12500..12519 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" primer_bind 12547..12566 /label=T7 /note="T7 promoter, forward primer" promoter 12547..12565 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 12576..13193 /codon_start=1 /gene="tetR from transposon Tn10" /product="tetracycline repressor TetR" /label=TetR /note="TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG" primer_bind complement(12649..12668) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" polyA_signal 13238..13359 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" enhancer 13414..13793 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 13794..13997 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 13948..13968 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 13999..14017 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 14020..14038 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 14048..14072 /label=LNCX /note="Human CMV promoter, forward primer" ORIGIN 1 agcttaccac catgtccgtc ctgacgccgc tgctgctgcg gggcttgaca ggctcggccc 61 ggcggctccc agtgccgcgc gccaagatcc attcgttgtc cgtcctgacg ccgctgctgc 121 tgcggggctt gacaggctcg gcccggcggc tcccagtgcc gcgcgccaag atccattcgt 181 tgggaggcag cgtgctcacc ccactcctgc tgcggggctt gacaggctcg gcccggcggc 241 tcccagtgcc gcgcgctaaa attcacagcc tctccgtcct gacgccgctg ctgctgcggg 301 gcttgacagg ctcggcccgg cggctcccag tgccgcgcgc caagatccat tcgttgggag 361 gcagcggagg cagcgagggc gggctggggc gtgctgtgtg cttgctgacc ggggcctccc 421 gcggcttcgg ccggacgctg gccccgctcc tggcctcgct gctgtcgccc ggctccgtgc 481 ttgtccttag cgcccgcaac gacgaggcac tgcgccagct ggaggccgag ctgggcgccg 541 agcggtctgg cctgcgcgtg gtgcgggtgc ccgccgacct gggcgccgag gccggcttgc 601 agcagctgct cggcgccctg cgcgagctcc cccggcccaa ggggctgcag cgactgctgc 661 ttatcaacaa cgcgggctct cttggggatg tgtccaaagg cttcgtggac ctgagtgact 721 ccactcaagt gaacaactac tgggcactga acttgacctc catgctctgc ctgacttcca 781 gcgtcctgaa ggccttcccg gacagtcctg gcctcaacag aaccgtggtt aacatctcgt 841 ccctctgtgc cctgcaacct ttcaaaggct gggcgctgta ctgtgcagga aaggctgctc 901 gtgatatgct gttccaggtc ctggcgctgg aggaacctaa tgtgagggtg ctgaactatg 961 ccccaggtcc tctggacaca gacatgcagc agttggcccg ggagacctcc gtggacccag 1021 acatgcgaaa agggctgcag gagctgaagg caaaggggaa gctggtggat tgcaaggtgt 1081 cagcccagaa actgctgagc ttactggaaa aggacgagtt caagtctgga gcccacgtgg 1141 acttctatga caaagaattc ggatccgaaa tcggtactgg ctttccattc gacccccatt 1201 atgtggaagt cctgggcgag cgcatgcact acgtcgatgt tggtccgcgc gatggcaccc 1261 ctgtgctgtt cctgcacggt aacccgacct cctcctacgt gtggcgcaac atcatcccgc 1321 atgttgcacc gacccatcgc tgcattgctc cagacctgat cggtatgggc aaatccgaca 1381 aaccagacct gggttatttc ttcgacgacc acgtccgctt catggatgcc ttcatcgaag 1441 ccctgggtct ggaagaggtc gtcctggtca ttcacgactg gggctccgct ctgggtttcc 1501 actgggccaa gcgcaatcca gagcgcgtca aaggtattgc atttatggag ttcatccgcc 1561 ctatcccgac ctgggacgaa tggccagaat ttgcccgcga gaccttccag gccttccgca 1621 ccaccgacgt cggccgcaag ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc 1681 cgatgggtgt cgtccgcccg ctgactgaag tcgagatgga ccattaccgc gagccgttcc 1741 tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa cgagctgcca atcgccggtg 1801 agccagcgaa catcgtcgcg ctggtcgaag aatacatgga ctggctgcac cagtcccctg 1861 tcccgaagct gctgttctgg ggcaccccag gcgttctgat cccaccggcc gaagccgctc 1921 gcctggccaa aagcctgcct aactgcaagg ctgtggacat cggcccgggt ctgaatctgc 1981 tgcaagaaga caacccggac ctgatcggca gcgagatcgc gcgctggctg tctactctgg 2041 agatttccgg tagcggccgc cctcctcctc cgccaccgcc accaccgccg ccgccgccac 2101 caccacctcc tccgcctcct ccaccaccac cgccaccgcc tccaccacct ggcggccgct 2161 ctcggtctct cgagatggat aaagactgcg aaatgaaacg cactaccctg gacagcccgc 2221 tgggtaaact ggaactgagc ggttgcgagc agggcctgca cgaaatcatt ttcctgggca 2281 aaggtacgag cgctgcggat gcggtcgaag taccggctcc ggctgcggtt ctgggtggcc 2341 cggagccgct gatgcaggct accgcatggc tgaacgcata ttttcaccag ccagaggcga 2401 ttgaagagtt ccctgtaccg gccctgcatc acccggtatt tcagcaggaa tcctttaccc 2461 gtcaggtact gtggaaactg ctgaaagtag tgaaattcgg cgaggtgatc tcctattccc 2521 atctggcggc gctggctggc aacccagcgg caactgcggc ggtgaaaacc gctctgtctg 2581 gtaatccggt gccaattctg atcccgtgcc accgtgtggt tcagggcgac ctggacgtag 2641 gtggctacga aggcggcctg gctgtgaaag aatggctgct ggcgcacgaa ggtcaccgtc 2701 tgggtaaacc aggcctgggc taaagctagc ggcgcgcccg gccgctcgag tctagagggc 2761 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 2821 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 2881 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 2941 tggggcagga cagcaagggg gaggattggg aagacaatag caggctagag gatcgatccc 3001 cgccccggac gaactaaacc tgactacgac atctctgccc cttcttcgcg gggcagtgca 3061 tgtaatccct tcagttggtt ggtacaactt gccaactggg ccctgttcca catgtgacac 3121 ggggggggac caaacacaaa ggggttctct gactgtagtt gacatcctta taaatggatg 3181 tgcacatttg ccaacactga gtggctttca tcctggagca gactttgcag tctgtggact 3241 gcaacacaac attgccttta tgtgtaactc ttggctgaag ctcttacacc aatgctgggg 3301 gacatgtacc tcccaggggc ccaggaagac tacgggaggc tacaccaacg tcaatcagag 3361 gggcctgtgt agctaccgat aagcggaccc tcaagagggc attagcaata gtgtttataa 3421 ggcccccttg ttaaccctaa acgggtagca tatgcttccc gggtagtagt atatactatc 3481 cagactaacc ctaattcaat agcatatgtt acccaacggg aagcatatgc tatcgaatta 3541 gggttagtaa aagggtccta aggaacagcg atatctccca ccccatgagc tgtcacggtt 3601 ttatttacat ggggtcagga ttccacgagg gtagtgaacc attttagtca caagggcagt 3661 ggctgaagat caaggagcgg gcagtgaact ctcctgaatc ttcgcctgct tcttcattct 3721 ccttcgttta gctaatagaa taactgctga gttgtgaaca gtaaggtgta tgtgaggtgc 3781 tcgaaaacaa ggtttcaggt gacgccccca gaataaaatt tggacggggg gttcagtggt 3841 ggcattgtgc tatgacacca atataaccct cacaaacccc ttgggcaata aatactagtg 3901 taggaatgaa acattctgaa tatctttaac aatagaaatc catggggtgg ggacaagccg 3961 taaagactgg atgtccatct cacacgaatt tatggctatg ggcaacacat aatcctagtg 4021 caatatgata ctggggttat taagatgtgt cccaggcagg gaccaagaca ggtgaaccat 4081 gttgttacac tctatttgta acaaggggaa agagagtgga cgccgacagc agcggactcc 4141 actggttgtc tctaacaccc ccgaaaatta aacggggctc cacgccaatg gggcccataa 4201 acaaagacaa gtggccactc ttttttttga aattgtggag tgggggcacg cgtcagcccc 4261 cacacgccgc cctgcggttt tggactgtaa aataagggtg taataacttg gctgattgta 4321 accccgctaa ccactgcggt caaaccactt gcccacaaaa ccactaatgg caccccgggg 4381 aatacctgca taagtaggtg ggcgggccaa gataggggcg cgattgctgc gatctggagg 4441 acaaattaca cacacttgcg cctgagcgcc aagcacaggg ttgttggtcc tcatattcac 4501 gaggtcgctg agagcacggt gggctaatgt tgccatgggt agcatatact acccaaatat 4561 ctggatagca tatgctatcc taatctatat ctgggtagca taggctatcc taatctatat 4621 ctgggtagca tatgctatcc taatctatat ctgggtagta tatgctatcc taatttatat 4681 ctgggtagca taggctatcc taatctatat ctgggtagca tatgctatcc taatctatat 4741 ctgggtagta tatgctatcc taatctgtat ccgggtagca tatgctatcc taatagagat 4801 tagggtagta tatgctatcc taatttatat ctgggtagca tatactaccc aaatatctgg 4861 atagcatatg ctatcctaat ctatatctgg gtagcatatg ctatcctaat ctatatctgg 4921 gtagcatagg ctatcctaat ctatatctgg gtagcatatg ctatcctaat ctatatctgg 4981 gtagtatatg ctatcctaat ttatatctgg gtagcatagg ctatcctaat ctatatctgg 5041 gtagcatatg ctatcctaat ctatatctgg gtagtatatg ctatcctaat ctgtatccgg 5101 gtagcatatg ctatcctcat gcatatacag tcagcatatg atacccagta gtagagtggg 5161 agtgctatcc tttgcatatg ccgccacctc ccaagggggc gtgaattttc gctgcttgtc 5221 cttttcctgc tggttgctcc cattcttagg tgaatttaag gaggccaggc taaagccgtc 5281 gcatgtctga ttgctcacca ggtaaatgtc gctaatgttt tccaacgcga gaaggtgttg 5341 agcgcggagc tgagtgacgt gacaacatgg gtatgcccaa ttgccccatg ttgggaggac 5401 gaaaatggtg acaagacaga tggccagaaa tacaccaaca gcacgcatga tgtctactgg 5461 ggatttattc tttagtgcgg gggaatacac ggcttttaat acgattgagg gcgtctccta 5521 acaagttaca tcactcctgc ccttcctcac cctcatctcc atcacctcct tcatctccgt 5581 catctccgtc atcaccctcc gcggcagccc cttccaccat aggtggaaac cagggaggca 5641 aatctactcc atcgtcaaag ctgcacacag tcaccctgat attgcaggta ggagcgggct 5701 ttgtcataac aaggtcctta atcgcatcct tcaaaacctc agcaaatata tgagtttgta 5761 aaaagaccat gaaataacag acaatggact cccttagcgg gccaggttgt gggccgggtc 5821 caggggccat tccaaagggg agacgactca atggtgtaag acgacattgt ggaatagcaa 5881 gggcagttcc tcgccttagg ttgtaaaggg aggtcttact acctccatat acgaacacac 5941 cggcgaccca agttccttcg tcggtagtcc tttctacgtg actcctagcc aggagagctc 6001 ttaaaccttc tgcaatgttc tcaaatttcg ggttggaacc tccttgacca cgatgctttt 6061 ccaaaccacc ctcctttttt gcgccctgcc tccatcaccc tgaccccggg gtccagtgct 6121 tgggccttct cctgggtcat ctgcggggcc ctgctctatc gctcccgggg gcacgtcagg 6181 ctcaccatct gggccacctt cttggtggta ttcaaaataa tcggcttccc ctacagggtg 6241 gaaaaatggc cttctacctg gagggggcct gcgcggtgga gacccggatg atgatgactg 6301 actactggga ctcctgggcc tcttttctcc acgtccacga cctctccccc tggctctttc 6361 acgacttccc cccctggctc tttcacgtcc tctaccccgg cggcctccac tacctcctcg 6421 accccggcct ccactacctc ctcgaccccg gcctccactg cctcctcgac cccggcctcc 6481 acctcctgct cctgcccctc ctgctcctgc ccctcctcct gctcctgccc ctcctgcccc 6541 tcctgctcct gcccctcctg cccctcctgc tcctgcccct cctgcccctc ctgctcctgc 6601 ccctcctgcc cctcctcctg ctcctgcccc tcctgcccct cctcctgctc ctgcccctcc 6661 tgcccctcct gctcctgccc ctcctgcccc tcctgctcct gcccctcctg cccctcctgc 6721 tcctgcccct cctgctcctg cccctcctgc tcctgcccct cctgctcctg cccctcctgc 6781 ccctcctgcc cctcctcctg ctcctgcccc tcctgctcct gcccctcctg cccctcctgc 6841 ccctcctgct cctgcccctc ctcctgctcc tgcccctcct gcccctcctg cccctcctcc 6901 tgctcctgcc cctcctgccc ctcctcctgc tcctgcccct cctcctgctc ctgcccctcc 6961 tgcccctcct gcccctcctc ctgctcctgc ccctcctgcc cctcctcctg ctcctgcccc 7021 tcctcctgct cctgcccctc ctgcccctcc tgcccctcct cctgctcctg cccctcctcc 7081 tgctcctgcc cctcctgccc ctcctgcccc tcctgcccct cctcctgctc ctgcccctcc 7141 tcctgctcct gcccctcctg ctcctgcccc tcccgctcct gctcctgctc ctgttccacc 7201 gtgggtccct ttgcagccaa tgcaacttgg acgtttttgg ggtctccgga caccatctct 7261 atgtcttggc cctgatcctg agccgcccgg ggctcctggt cttccgcctc ctcgtcctcg 7321 tcctcttccc cgtcctcgtc catggttatc accccctctt ctttgaggtc cactgccgcc 7381 ggagccttct ggtccagatg tgtctccctt ctctcctagg ccatttccag gtcctgtacc 7441 tggcccctcg tcagacatga ttcacactaa aagagatcaa tagacatctt tattagacga 7501 cgctcagtga atacagggag tgcagactcc tgccccctcc aacagccccc ccaccctcat 7561 ccccttcatg gtcgctgtca gacagatcca ggtctgaaaa ttccccatcc tccgaaccat 7621 cctcgtcctc atcaccaatt actcgcagcc cggaaaactc ccgctgaaca tcctcaagat 7681 ttgcgtcctg agcctcaagc caggcctcaa attcctcgtc cccctttttg ctggacggta 7741 gggatgggga ttctcgggac ccctcctctt cctcttcaag gtcaccagac agagatgcta 7801 ctggggcaac ggaagaaaag ctgggtgcgg cctgtgagga tcagcttatc gatgataagc 7861 tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt 7921 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 7981 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 8041 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 8101 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 8161 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 8221 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 8281 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca 8341 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 8401 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 8461 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 8521 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 8581 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac 8641 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 8701 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 8761 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 8821 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 8881 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 8941 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 9001 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 9061 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 9121 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 9181 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 9241 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 9301 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 9361 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 9421 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 9481 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 9541 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 9601 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 9661 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 9721 ctttttacgg ttcctggcct tttgctggcc ttgaagctgt ccctgatggt cgtcatctac 9781 ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat 9841 cataatgggg aaggccatcc agcctcgcgt cgaactagat gatccggctg tggaatgtgt 9901 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc 9961 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta 10021 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc 10081 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta 10141 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct 10201 tttttggagg gtgaccgcca cgaccggtgc cgccaccatc ccctgaccca cgcccctgac 10261 ccctcacaag gagacgacct tccatgaccg agtacaagcc cacggtgcgc ctcgccaccc 10321 gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca 10381 cgcgccacac cgtcgacccc gaccgccaca tcgaacgcgt caccgagctg caagaactct 10441 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg 10501 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc 10561 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc 10621 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg 10681 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc 10741 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc 10801 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca 10861 tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga 10921 gcgcacgacc cggtccgacg cggcccgacg ggtccgaggg gggtcgacct cgaaacttgt 10981 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 11041 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 11101 tctggatctg atccgaaccc cttcctcgac caattctcat gtttgacagc ttatcatcgc 11161 agatccgggc aacgttgttg ccattgctcg accaattctc atgtttgaca gcttatcatc 11221 gcagatccgt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 11281 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta 11341 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg 11401 cgctgcttcg cgatgtacgg gccagatata cgcgtatctg aggggactag ggtgtgttta 11461 ggcgaaaagc ggggcttcgg ttgtacgcgg ttaggagtcc cctcaggata tagtagtttc 11521 gcttttgcat agggaggggg aaatgtagtc ttatgcaata ctcttgtagt cttgcaacat 11581 ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt 11641 ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg 11701 gattggacga accactgaat tccgcattgc agagatattg tatttaagtg cctagctcga 11761 tacaataaac gccatttgac cattcaccac attggtgtgc acctccaagc tggccgggga 11821 tcctctaggg cctctgagct attccagaag tagtgaagag gcttttttgg aggcctaggc 11881 ttttgcaaaa agctccggat cgatcctgag aacttcaggg tgagtttggg gacccttgat 11941 tgttctttct ttttcgctat tgtaaaattc atgttatatg gagggggcaa agttttcagg 12001 gtgttgttta gaatgggaag atgtcccttg tatcaccatg gaccctcatg ataattttgt 12061 ttctttcact ttctactctg ttgacaacca ttgtctcctc ttattttctt ttcattttct 12121 gtaacttttt cgttaaactt tagcttgcat ttgtaacgaa tttttaaatt cacttttgtt 12181 tatttgtcag attgtaagta ctttctctaa tcactttttt ttcaaggcaa tcagggtata 12241 ttatattgta cttcagcaca gttttagaga acaattgtta taattaaatg ataaggtaga 12301 atatttctgc atataaattc tggctggcgt ggaaatattc ttattggtag aaacaactac 12361 atcctggtca tcatcctgcc tttctcttta tggttacaat gatatacact gtttgagatg 12421 aggataaaat actctgagtc caaaccgggc ccctctgcta accatgttca tgccttcttc 12481 tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa 12541 gaattgtaat acgactcact atagggcgaa ttgatatgtc tagattagat aaaagtaaag 12601 tgattaacag cgcattagag ctgcttaatg aggtcggaat cgaaggttta acaacccgta 12661 aactcgccca gaagctaggt gtagagcagc ctacattgta ttggcatgta aaaaataagc 12721 gggctttgct cgacgcctta gccattgaga tgttagatag gcaccatact cacttttgcc 12781 ctttagaagg ggaaagctgg caagattttt tacgtaataa cgctaaaagt tttagatgtg 12841 ctttactaag tcatcgcgat ggagcaaaag tacatttagg tacacggcct acagaaaaac 12901 agtatgaaac tctcgaaaat caattagcct ttttatgcca acaaggtttt tcactagaga 12961 atgcattata tgcactcagc gctgtggggc attttacttt aggttgcgta ttggaagatc 13021 aagagcatca agtcgctaaa gaagaaaggg aaacacctac tactgatagt atgccgccat 13081 tattacgaca agctatcgaa ttatttgatc accaaggtgc agagccagcc ttcttattcg 13141 gccttgaatt gatcatatgc ggattagaaa aacaacttaa atgtgaaagt gggtccgcgt 13201 acagcggatc ccgggaattc agatcttatt aaagcagaac ttgtttattg cagcttataa 13261 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 13321 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggt cgaatccatg 13381 gcgcagaact ggtaggtatg gaagatccgc gttgacattg attattgact agttattaat 13441 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 13501 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 13561 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 13621 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 13681 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 13741 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 13801 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 13861 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 13921 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 13981 tctatataag cagagctctc cctatcagtg atagagatct ccctatcagt gatagagatc 14041 gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg 14101 acctccatag aagacaccgg gaccgatcca gcctccggac tctagcgttt aaactta //