LOCUS Exported 9176 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION gRNA_B to knockout human AMPK alpha 2 using Cas9n. ACCESSION . VERSION . KEYWORDS pX462-hPRKAA2-gRNA_B SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9176) AUTHORS Toyama EQ, Herzig S, Courchet J, Lewis TL Jr, Loson OC, Hellberg K, Young NP, Chen H, Polleux F, Chan DC, Shaw RJ TITLE Metabolism. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress. JOURNAL Science. 2016 Jan 15;351(6270):275-81. doi: 10.1126/science.aab4138. PUBMED 26816379 REFERENCE 2 (bases 1 to 9176) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9176 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 270..345 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 442..727 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" gap 841..852 /estimated_length=12 intron 1007..1234 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1246..1255 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1255..1320 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1327..1347 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1372..5472 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5473..5520 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5536..5589 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 5590..6189 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(5590..5609) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind complement(6217..6234) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6223..6430 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 6439..6579 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 6439..6568 /label=AAV2 ITR rep_origin 6654..7109 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6741..6760) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6951..6972 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7126..7145) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7245..7267 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7305..7323) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7391..7495 /gene="bla" /label=AmpR promoter CDS 7496..8356 /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(7714..7733) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8527..9115 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9016..9035 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg aagatcggac actacgtgcg ttttagagct agaaatagca agttaaaata 301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 841 nnnnnnnnnn nncgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 901 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 961 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg 1021 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 1081 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 1141 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta 1201 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat 1261 aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga tgacgataag 1321 atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag 1381 tacagcatcg gcctggccat cggcaccaac tctgtgggct gggccgtgat caccgacgag 1441 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag 1501 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg 1561 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag 1621 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc 1681 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac 1741 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac 1801 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc 1861 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg 1921 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc 1981 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat 2041 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggcaacct gattgccctg 2101 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg 2161 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac 2221 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac 2281 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga 2341 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct 2401 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac 2461 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac 2521 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc 2581 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg 2641 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg 2701 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg 2761 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag 2821 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac 2881 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg 2941 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag 3001 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg 3061 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa 3121 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag 3181 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca 3241 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac 3301 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg 3361 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag 3421 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt 3481 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt 3541 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg 3601 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc 3661 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc 3721 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc 3781 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg 3841 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag 3901 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg 3961 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg 4021 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag 4081 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc 4141 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac 4201 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc 4261 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc 4321 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg 4381 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag 4441 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac 4501 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag 4561 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 4621 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 4681 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag 4741 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg 4801 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg 4861 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc 4921 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc 4981 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac 5041 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag 5101 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac 5161 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac 5221 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag 5281 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc 5341 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac 5401 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag 5461 ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag 5521 gaattcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat 5581 cctggcccaa tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc 5641 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc 5701 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc 5761 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc 5821 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag 5881 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg 5941 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag 6001 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc 6061 gccttcctgg agacctccgc gccccacaac ctccccttct acgagcggct cggcttcacc 6121 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc 6181 ggtgcctgag aattctaact agagctcgct gatcagcctc gactgtgcct tctagttgcc 6241 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 6301 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 6361 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc 6421 atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg 6481 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg 6541 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc 6601 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc 6661 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 6721 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 6781 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 6841 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc 6901 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 6961 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat 7021 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 7081 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga 7141 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc 7201 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 7261 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct 7321 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg 7381 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 7441 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 7501 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 7561 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 7621 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 7681 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 7741 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 7801 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 7861 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 7921 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 7981 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 8041 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 8101 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 8161 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg 8221 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 8281 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 8341 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 8401 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 8461 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 8521 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 8581 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 8641 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca 8701 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 8761 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 8821 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 8881 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 8941 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 9001 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 9061 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 9121 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt //