LOCUS Exported 9476 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses FokI-dCas9 and gRNA. ACCESSION . VERSION . KEYWORDS pX330A_FokI-1x3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9476) AUTHORS Nakagawa Y, Sakuma T, Sakamoto T, Ohmuraya M, Nakagata N, Yamamoto T TITLE Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. JOURNAL BMC Biotechnol. 2015 May 22;15(1):33. PUBMED 25997509 REFERENCE 2 (bases 1 to 9476) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9476 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 8..293 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 295..572 /label=chicken beta-actin promoter intron 573..801 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 822..842 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 849..1436 /codon_start=1 /product="nonspecific DNA cleavage domain of the FokI endonuclease (Li et al., 1992)" /label=FokI cleavage domain /note="must dimerize to be catalytically active" /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF" CDS 1452..5552 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5553..5600 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5628..5645) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5634..5841 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5850..5990 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5850..5979 /label=AAV2 ITR rep_origin 6065..6520 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6152..6171) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6362..6383 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6537..6556) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6656..6678 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6716..6734) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6802..6906 /gene="bla" /label=AmpR promoter CDS 6907..7767 /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(7125..7144) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7938..8526 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8427..8446 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8588..8828 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8588..8608 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8759..8778 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8855..8930 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" CDS complement(9016..9321) /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL" primer_bind 9171..9193 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 9185..9202 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 9186..9202 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 9212..9231 /label=T7 /note="T7 promoter, forward primer" promoter 9212..9230 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(9278..9298) /label=T3 /note="T3 promoter, forward primer" promoter complement(9278..9296) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(9317..9333) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9317..9333) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(9330..9352) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 9341..9357 /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(9365..9395) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9410..9431 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." ORIGIN 1 aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat 241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg 301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg 421 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg 481 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc 541 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc 601 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 661 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg 721 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg 781 cctgaaatca ctttttttca ggttggaccg gtgccaccat gcctaagaag aagcggaagg 841 tgagctccca actcgtgaag agtgaacttg aggagaaaaa gtcggagctg cggcacaaat 901 tgaaatacgt accgcatgaa tacatcgaac ttatcgaaat tgctaggaac tcgactcaag 961 acagaatcct tgagatgaag gtaatggagt tctttatgaa ggtttatgga taccgaggga 1021 agcatctcgg tggatcacga aaacccgacg gagcaatcta tacggtgggg agcccgattg 1081 attacggagt gatcgtcgac acgaaagcct acagcggtgg gtacaatctt cccatcgggc 1141 aggcagatga gatgcaacgt tatgtcgaag aaaatcagac caggaacaaa cacatcaatc 1201 caaatgagtg gtggaaagtg tatccttcat cagtgaccga gtttaagttt ttgtttgtct 1261 ctgggcattt caaaggcaac tataaggccc agctcacacg gttgaatcac attacgaact 1321 gcaatggtgc ggttttgtcc gtagaggaac tgctcattgg tggagaaatg atcaaagcgg 1381 gaactctgac actggaagaa gtcagacgca agtttaacaa tggcgagatc aatttcggtg 1441 gcggtggatc cgacaagaag tacagcatcg gcctggccat cggcaccaac tctgtgggct 1501 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca 1561 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa 1621 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc 1681 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct 1741 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca 1801 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc 1861 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc 1921 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca 1981 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg 2041 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca 2101 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt 2161 tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc 2221 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc 2281 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg 2341 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga 2401 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc 2461 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg 2521 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc 2581 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc 2641 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag 2701 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg 2761 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg 2821 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact 2881 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact 2941 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact 3001 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg 3061 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga 3121 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg 3181 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc 3241 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag 3301 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa 3361 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg 3421 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga 3481 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc 3541 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg 3601 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc 3661 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga 3721 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc 3781 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag 3841 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga 3901 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg 3961 tggacgccat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga 4021 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga 4081 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg 4141 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca 4201 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc 4261 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc 4321 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga 4381 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga 4441 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg 4501 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct 4561 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc 4621 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc 4681 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga 4741 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata 4801 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca 4861 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga 4921 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc 4981 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc 5041 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg 5101 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc 5161 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt 5221 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca 5281 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc 5341 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc 5401 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca 5461 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga 5521 cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg 5581 gccaggcaaa aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc 5641 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 5701 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 5761 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 5821 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc 5881 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 5941 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat 6001 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca 6061 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 6121 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 6181 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 6241 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt 6301 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 6361 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat 6421 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 6481 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca 6541 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 6601 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 6661 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 6721 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt 6781 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 6841 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 6901 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 6961 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 7021 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 7081 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 7141 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 7201 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 7261 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 7321 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 7381 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 7441 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 7501 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 7561 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 7621 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 7681 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 7741 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 7801 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 7861 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 7921 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 7981 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 8041 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 8101 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 8161 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 8221 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 8281 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 8341 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 8401 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 8461 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 8521 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 8581 cacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt 8641 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg 8701 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga 8761 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg 8821 gaaaggacga aacaccgggt cttcgagaag acctgtttta gagctagaaa tagcaagtta 8881 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt 8941 tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt gcgccaattc 9001 tgcagacaaa tggctctaga gaccggcgcc gctacagggc gcgtcccatt cgccattcag 9061 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc 9121 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg 9181 acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg 9241 taccgggccc cccctcgagg tcctccagct tttgttccct ttagtgaggg ttaattgcgc 9301 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 9361 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 9421 aactcacatt aattgcgttg cgctcactgc ccgctttcca ccggtggtct ctctag //