质粒名称: | pC001 - huLshC2C2-MBP for bacterial expression |
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出品公司: | 爱迪基因 |
目录编号: | 79150 |
存储实验室: | Feng Zhang |
载体骨架基本信息 |
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载体名称: | BPV00356 pET21GG2-His(6)-MBP_Flag-Avi |
载体出品公司: | Zhang lab |
载体抗性: | 氨苄青霉素 |
空载体大小: | 6650 bp |
完整质粒大小: | 10955 bp |
载体修饰: | 人细胞色素c氧化酶亚基VIII(human cytochrome C oxidase subunit VIII) |
载体类型: | 哺乳细胞表达 |
筛选标记: | 新霉素(G418) |
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: | C2c2 |
物种来源: | -- |
插入大小: | 4305 bp |
基因突变: | -- |
GenBank编号 | -- |
启动子 | -- |
标签/融合蛋白: | 人细胞色素c氧化酶亚基VIII(N端) |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | AgeI(未破坏) |
3' 克隆位点: | NotI (未破坏) |
5' 测序引物 | CMV-F |
3' 测序引物 | SV40pA-R |
文献引用方法 |
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材料和方法部分: | pC001 - huLshC2C2-MBP for bacterial expression was a gift from Feng Zhang (Addgene plasmid # 79150 ; http://n2t.net/addgene:79150 ; RRID:Addgene_79150) |
参考文献部分: | C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F. Science. 2016 Jun 2. pii: aaf5573. 10.1126/science.aaf5573 PubMed 27256883 |
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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 pC001 - huLshC2C2-MBP for bacterial expression 10951 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Expresses human codon-optimized LshC2c2 for purification in E. coli. ACCESSION . VERSION . KEYWORDS pC001 - huLshC2C2-MBP for bacterial expression SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10951) AUTHORS Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F TITLE C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. JOURNAL Science. 2016 Jun 2. pii: aaf5573. PUBMED 27256883 REFERENCE 2 (bases 1 to 10951) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10951 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1012..1031 /label=LacI-R /note="LacI, reverse primer" promoter complement(1051..1128) /gene="lacI" /label=lacI promoter primer_bind 1334..1353 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1437..1456 /label=T7 /note="T7 promoter, forward primer" promoter 1437..1455 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1456..1480 /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)." RBS 1495..1517 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1531..1548 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" regulatory 2503..2512 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2665..2682 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 2689..2709 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2710..6876 /codon_start=1 /product="type VI CRISPR effector from Leptotrichia shahii with RNA-guided RNAse activity (Abudayyeh et al., 2016)" /label=LshC2c2 /translation="MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNN KEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEE VVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEY TNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYE EHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEK ILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKH EKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTE IFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIE IEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLE LITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFID NKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVS KALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFD TIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIEN YYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDI NDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEY QNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIFNNYY EDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKK DLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYY PKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEI DAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIR DLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAY PKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHF YIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLI GNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL" CDS 6877..6924 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 6931..7011 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" CDS 7035..7058 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 7059..7103 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 7119..7136 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(7189..7207) /label=T7 Term /note="T7 terminator, reverse primer" terminator 7203..7250 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 7287..7742 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7374..7393) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7584..7605 /label=F1ori-F /note="F1 origin, forward primer" promoter 7768..7872 /gene="bla" /label=AmpR promoter CDS 7873..8733 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(8091..8110) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8904..9492 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9393..9412 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 9646..9663 /label=L4440 /note="L4440 vector, forward primer" misc_feature 9678..9817 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(9743..9762) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 9903..9925 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(9919..10110) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(10919..1050) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" ORIGIN 1 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 61 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg 121 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg 181 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac 241 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag 301 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg 361 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg 421 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga 481 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac 541 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga 601 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg 661 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac 721 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc 781 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag 841 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg 901 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga 961 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc 1021 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg 1081 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac 1141 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga 1201 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca 1261 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc 1321 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg 1381 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat 1441 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt 1501 taactttaag aaggagatat acatatgggt catcatcatc accatcacaa aatcgaagaa 1561 ggcaaactgg ttatttggat caatggcgat aaaggctata atggtctggc agaagttggc 1621 aaaaaattcg aaaaagatac cggcattaaa gtgaccgttg aacatccgga taaactggaa 1681 gaaaaatttc cgcaggttgc agcaaccggt gatggtccgg atattatctt ttgggcacat 1741 gatcgttttg gtggttatgc acagagcggt ctgctggcag aaattacacc ggataaagca 1801 tttcaggaca aactgtatcc gtttacctgg gatgcagttc gctataacgg taaactgatt 1861 gcatatccga ttgcagttga agcactgagc ctgatctata acaaagatct gctgccgaat 1921 ccgcctaaaa cctgggaaga aattccggca ctggataaag aactgaaagc aaaaggtaaa 1981 agcgcactga tgtttaatct gcaagaaccg tattttacct ggcctctgat tgcagcagat 2041 ggtggctatg cattcaaata tgaaaacggc aaatatgata tcaaagacgt gggtgttgat 2101 aatgcgggtg caaaagccgg tctgaccttt ctggttgatc tgattaaaaa caaacacatg 2161 aacgccgata ccgattatag cattgcagaa gcagcattta acaaaggtga aaccgcaatg 2221 acaattaatg gtccgtgggc atggtcaaat attgatacca gcaaagtgaa ttatggtgtt 2281 accgttctgc cgacatttaa aggtcagccg agcaaaccgt ttgttggtgt tctgagcgca 2341 ggtattaatg cagcaagccc gaacaaagaa ctggcaaaag aatttctgga aaactatctg 2401 ctgaccgatg aaggtctgga agcagtgaat aaagataaac cgctgggtgc agttgcactg 2461 aaaagctatg aagaagaact ggttaaagat ccgcgtattg cagccaccat ggaaaatgca 2521 cagaaaggcg aaattatgcc gaatattccg cagatgagcg cattttggta tgccgttcgt 2581 accgcagtga ttaatgccgc atcaggtcgt cagaccgttg atgaagccct gaaagatgcc 2641 cagaccggtg caagcaccgg atccctggtt ccgcgtggta gcgctagcga aaatctgtat 2701 tttcagggaa tggggaacct gttcggacac aagcggtggt acgaggtgcg agataagaaa 2761 gacttcaaga tcaagcgaaa ggtgaaagtc aagcggaatt atgacgggaa caagtacatt 2821 ctgaacatca acgaaaacaa caacaaagag aagatcgaca acaacaagtt catcagaaag 2881 tacatcaact acaagaaaaa cgataatatt ctgaaggagt tcactaggaa atttcatgca 2941 ggaaatatcc tgttcaaact gaagggcaaa gaagggatca ttagaattga gaataacgac 3001 gacttcctgg agacagagga agtggtgctg tatatcgagg cctacggcaa gagcgagaag 3061 ctgaaagcac tggggatcac aaagaaaaag atcattgacg aggccatcag gcagggcatt 3121 actaaggacg ataaaaagat cgagatcaag cgacaggaga acgaggaaga gatcgaaatt 3181 gacatccggg atgagtacac taataagacc ctgaacgact gctccatcat tctgcgcatc 3241 attgaaaacg atgaactgga gacaaagaag agcatctacg agatcttcaa gaacatcaat 3301 atgagcctgt ataagatcat cgagaagatc atcgaaaacg agacagaaaa ggtgtttgaa 3361 aatagatact acgaagagca cctgagggag aagctgctga aagacgataa gattgacgtg 3421 atcctgacca acttcatgga aatccgggag aagatcaagt ctaatctgga gatcctgggc 3481 ttcgtgaagt tttacctgaa cgtcggcggg gacaaaaaga aaagtaaaaa taagaaaatg 3541 ctggtggaaa agattctgaa catcaatgtg gatctgaccg tcgaggacat tgccgatttc 3601 gtgatcaagg agctggaatt ttggaacatc acaaagcgca ttgagaaagt gaagaaagtc 3661 aataacgagt tcctggagaa gcggagaaat cggacataca tcaagtccta tgtgctgctg 3721 gacaagcacg aaaagtttaa aatcgagaga gaaaacaaga aggataagat cgtgaagttc 3781 tttgtcgaga acattaagaa caactctatc aaggaaaaga ttgagaagat cctggctgag 3841 ttcaagatcg acgagctgat taagaaactg gagaaggaac tgaagaaagg gaactgtgat 3901 accgagatct tcggaatctt taagaagcat tacaaggtga acttcgacag caagaaattt 3961 tccaagaaat ctgatgaaga gaaggagctg tataagatca tctacagata cctgaagggc 4021 agaattgaaa aaatcctggt gaacgagcag aaggtcagac tgaagaaaat ggagaagatc 4081 gagatcgaaa agattctgaa tgaaagtatc ctgtcagaga aaattctgaa gagagtgaaa 4141 cagtatacac tggagcacat tatgtacctg gggaagctga ggcataacga catcgatatg 4201 accacagtga atactgacga tttcagccgc ctgcacgcca aggaagagct ggacctggaa 4261 ctgatcacct tctttgccag cacaaatatg gagctgaaca agatcttttc ccgagaaaac 4321 atcaacaacg acgagaacat cgatttcttt ggaggcgacc gggagaagaa ctatgtgctg 4381 gataagaaaa tcctgaatag taagatcaag atcatccgcg acctggattt catcgataac 4441 aagaacaaca tcacaaacaa cttcattcga aagtttacaa agatcggcac taatgaaagg 4501 aaccgcatcc tgcatgccat ttccaaagag agggacctgc aggggactca ggacgattac 4561 aacaaagtga tcaacatcat tcagaatctg aagatctccg atgaagaggt gagcaaagct 4621 ctgaacctgg acgtggtctt taaggacaag aaaaacatca tcacaaagat caatgacatc 4681 aagatctctg aagagaacaa caacgatatc aagtatctgc ccagcttcag caaagtgctg 4741 cccgaaatcc tgaacctgta ccgcaacaat cccaagaatg agccttttga cacaatcgag 4801 actgaaaaaa ttgtgctgaa cgctctgatc tacgtcaata aggagctgta taagaaactg 4861 atcctggagg acgatctgga agagaacgag tccaagaata tcttcctgca ggaactgaag 4921 aaaaccctgg gcaacattga cgaaatcgat gagaacatca tcgagaacta ctacaagaac 4981 gcacagattt ctgccagtaa ggggaacaac aaggcaatca agaaatatca gaagaaagtg 5041 atcgagtgct acattggata tctgcgcaaa aactacgaag agctgttcga cttttcagac 5101 ttcaagatga acatccagga aatcaagaaa cagattaagg acatcaacga taacaagact 5161 tatgagcgga tcaccgtgaa aaccagcgac aaaaccattg tcatcaacga cgatttcgag 5221 tacatcattt ctatctttgc actgctgaac agtaatgccg tgattaataa gatccgaaac 5281 agattcttcg ccaccagcgt gtggctgaac acctcagaat accagaatat cattgacatc 5341 ctggatgaga ttatgcagct gaataccctg cggaacgaat gcatcacaga gaactggaat 5401 ctgaacctgg aagagttcat tcagaagatg aaagagatcg aaaaggattt cgacgacttc 5461 aagatccaga ctaagaaaga aatcttcaac aactactacg aggacatcaa gaacaacatt 5521 ctgaccgagt ttaaagacga tatcaacggc tgtgatgtgc tggaaaagaa actggagaag 5581 attgtcatct tcgacgatga aaccaagttc gagatcgaca agaaatccaa catcctgcag 5641 gatgaacaga gaaagctgtc taacatcaac aagaaggacc tgaagaaaaa ggtggatcag 5701 tatatcaagg acaaagatca ggagatcaag tctaaaatcc tgtgcaggat cattttcaac 5761 agtgactttc tgaaaaagta caagaaggaa atcgacaatc tgattgagga tatggagtct 5821 gaaaatgaga acaagttcca ggagatctac tatcccaagg aacggaagaa cgagctgtat 5881 atctacaaaa agaatctgtt cctgaacatc ggaaatccta actttgacaa gatctacggc 5941 ctgattagca acgacatcaa gatggccgat gctaaattcc tgtttaatat cgatggaaag 6001 aacatcagaa agaacaaaat cagtgagatc gacgctattc tgaagaatct gaacgataaa 6061 ctgaacggct actcaaagga atacaaggag aagtacatca aaaagctgaa ggagaacgac 6121 gatttctttg caaagaacat ccagaataag aactacaaat ccttcgaaaa ggactataac 6181 cgcgtgtctg agtacaaaaa gattcgagat ctggtcgagt tcaactatct gaacaaaatc 6241 gagtcctacc tgattgacat caactggaag ctggctattc agatggcaag attcgaaagg 6301 gatatgcact atatcgtgaa tggactgagg gagctgggca tcattaagct gtcaggctat 6361 aacaccggga tcagcagggc atacccaaag cgcaatggaa gcgacggctt ttacactacc 6421 acagcctact acaagttctt tgatgaagag tcctacaaga agttcgagaa gatttgctac 6481 gggtttggaa tcgacctgag cgaaaattcc gagatcaaca agcctgaaaa tgagagcatt 6541 cggaactata tctcccattt ctacatcgtg agaaatccat ttgccgacta cagtattgct 6601 gagcagatcg atcgggtgag caacctgctg tcatatagca cacgctacaa caattcaact 6661 tatgccagcg tgttcgaagt ctttaaaaag gacgtgaatc tggactacga tgagctgaaa 6721 aagaaattca aactgatcgg caacaatgat attctggagc gcctgatgaa gcccaagaaa 6781 gtgagcgtcc tggaactgga gtcctacaac agtgactaca ttaagaatct gatcattgaa 6841 ctgctgacca aaatcgagaa tactaacgat accctgaaaa ggccggcggc cacgaaaaag 6901 gccggccagg caaaaaagaa aaagggatcc tacccatacg atgttccaga ttacgcttat 6961 ccctacgacg tgcctgatta tgcataccca tatgatgtcc ccgactatgc ctaagcctcg 7021 agggtggtgg tagcgattat aaagatgatg atgataaagg cctgaacgat atctttgaag 7081 cccagaaaat tgaatggcac gagtaaaagc ttgtcgagca ccaccaccac caccactgag 7141 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat 7201 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag 7261 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg 7321 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 7381 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 7441 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 7501 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 7561 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 7621 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 7681 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat 7741 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 7801 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 7861 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 7921 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 7981 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 8041 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 8101 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 8161 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 8221 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 8281 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 8341 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 8401 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta 8461 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 8521 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 8581 gagcgtggta gtcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 8641 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 8701 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 8761 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 8821 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 8881 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 8941 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 9001 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 9061 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 9121 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 9181 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 9241 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 9301 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 9361 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 9421 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 9481 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 9541 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 9601 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 9661 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 9721 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata 9781 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc 9841 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 9901 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct 9961 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc 10021 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat 10081 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt 10141 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga 10201 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg 10261 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt 10321 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc 10381 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc 10441 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc 10501 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac 10561 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg 10621 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc 10681 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca 10741 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc 10801 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg 10861 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc 10921 actgcccgct ttccagtcgg gaaacctgtc g //