LOCUS Exported 9059 bp ds-DNA circular SYN 05-JAN-2023 DEFINITION Bicistronic vector expressing CMV promoter driven AsCpf1, and U6 promoter driven guide RNA that target hINS. ACCESSION . VERSION . KEYWORDS CMV:AsCpf1-2A-GFP-U6-INS-sg SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9059) TITLE Multiplex genome editing of human pluripotent stem cells using Cpf1 REFERENCE 2 (bases 1 to 9059) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 5, 2023 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9059 /organism="synthetic DNA construct" /mol_type="other DNA" intron 127..354 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 370..379 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 382..402 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 427..4344 /codon_start=1 /product="CRISPR-associated protein from Acidaminococcus sp. BV3L6 (Zetsche et al., 2015)" /label=AsCpf1 /translation="TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNA IHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDK FTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLN LAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLR NENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGK ITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLK KQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKAR NYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKA LSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPL EITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDL SSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKD QKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFH VPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQF TSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDV KTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENH RFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMR NSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESK DLKLQNGISNQDWLAYIQELRN" regulatory 4064..4073 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4345..4392 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 4408..4461 /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 4462..5175 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 4462..5175 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(4504..4525) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4765..4784) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 5112..5133 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(5203..5220) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5209..5416 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5425..5565 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5425..5554 /label=AAV2 ITR rep_origin 5640..6095 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5727..5746) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5937..5958 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6112..6131) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6231..6253 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6291..6309) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6377..6481 /gene="bla" /label=AmpR promoter CDS 6482..7342 /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(6700..6719) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7513..8101 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8002..8021 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8163..8403 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8163..8183 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8334..8353 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8463..8538 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 8635..8920 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" gap 9039..9055 /estimated_length=17 ORIGIN 1 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga 61 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg 121 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc 181 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct 241 tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt 301 ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga 361 ccggtggccg ccaccatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca 421 gcagccacac agttcgaggg ctttaccaac ctgtatcagg tgagcaagac actgcggttt 481 gagctgatcc cacagggcaa gaccctgaag cacatccagg agcagggctt catcgaggag 541 gacaaggccc gcaatgatca ctacaaggag ctgaagccca tcatcgatcg gatctacaag 601 acctatgccg accagtgcct gcagctggtg cagctggatt gggagaacct gagcgccgcc 661 atcgactcct atagaaagga gaaaaccgag gagacaagga acgccctgat cgaggagcag 721 gccacatatc gcaatgccat ccacgactac ttcatcggcc ggacagacaa cctgaccgat 781 gccatcaata agagacacgc cgagatctac aagggcctgt tcaaggccga gctgtttaat 841 ggcaaggtgc tgaagcagct gggcaccgtg accacaaccg agcacgagaa cgccctgctg 901 cggagcttcg acaagtttac aacctacttc tccggctttt atgagaacag gaagaacgtg 961 ttcagcgccg aggatatcag cacagccatc ccacaccgca tcgtgcagga caacttcccc 1021 aagtttaagg agaattgtca catcttcaca cgcctgatca ccgccgtgcc cagcctgcgg 1081 gagcactttg agaacgtgaa gaaggccatc ggcatcttcg tgagcacctc catcgaggag 1141 gtgttttcct tcccttttta taaccagctg ctgacacaga cccagatcga cctgtataac 1201 cagctgctgg gaggaatctc tcgggaggca ggcaccgaga agatcaaggg cctgaacgag 1261 gtgctgaatc tggccatcca gaagaatgat gagacagccc acatcatcgc ctccctgcca 1321 cacagattca tccccctgtt taagcagatc ctgtccgata ggaacaccct gtctttcatc 1381 ctggaggagt ttaagagcga cgaggaagtg atccagtcct tctgcaagta caagacactg 1441 ctgagaaacg agaacgtgct ggagacagcc gaggccctgt ttaacgagct gaacagcatc 1501 gacctgacac acatcttcat cagccacaag aagctggaga caatcagcag cgccctgtgc 1561 gaccactggg atacactgag gaatgccctg tatgagcgga gaatctccga gctgacaggc 1621 aagatcacca agtctgccaa ggagaaggtg cagcgcagcc tgaagcacga ggatatcaac 1681 ctgcaggaga tcatctctgc cgcaggcaag gagctgagcg aggccttcaa gcagaaaacc 1741 agcgagatcc tgtcccacgc acacgccgcc ctggatcagc cactgcctac aaccctgaag 1801 aagcaggagg agaaggagat cctgaagtct cagctggaca gcctgctggg cctgtaccac 1861 ctgctggact ggtttgccgt ggatgagtcc aacgaggtgg accccgagtt ctctgcccgg 1921 ctgaccggca tcaagctgga gatggagcct tctctgagct tctacaacaa ggccagaaat 1981 tatgccacca agaagcccta ctccgtggag aagttcaagc tgaactttca gatgcctaca 2041 ctggcctctg gctgggacgt gaataaggag aagaacaatg gcgccatcct gtttgtgaag 2101 aacggcctgt actatctggg catcatgcca aagcagaagg gcaggtataa ggccctgagc 2161 ttcgagccca cagagaaaac cagcgagggc tttgataaga tgtactatga ctacttccct 2221 gatgccgcca agatgatccc aaagtgcagc acccagctga aggccgtgac agcccacttt 2281 cagacccaca caacccccat cctgctgtcc aacaatttca tcgagcctct ggagatcaca 2341 aaggagatct acgacctgaa caatcctgag aaggagccaa agaagtttca gacagcctac 2401 gccaagaaaa ccggcgacca gaagggctac agagaggccc tgtgcaagtg gatcgacttc 2461 acaagggatt ttctgtccaa gtataccaag acaacctcta tcgatctgtc tagcctgcgg 2521 ccatcctctc agtataagga cctgggcgag tactatgccg agctgaatcc cctgctgtac 2581 cacatcagct tccagagaat cgccgagaag gagatcatgg atgccgtgga gacaggcaag 2641 ctgtacctgt tccagatcta taacaaggac tttgccaagg gccaccacgg caagcctaat 2701 ctgcacacac tgtattggac cggcctgttt tctccagaga acctggccaa gacaagcatc 2761 aagctgaatg gccaggccga gctgttctac cgccctaagt ccaggatgaa gaggatggca 2821 caccggctgg gagagaagat gctgaacaag aagctgaagg atcagaaaac cccaatcccc 2881 gacaccctgt accaggagct gtacgactat gtgaatcaca gactgtccca cgacctgtct 2941 gatgaggcca gggccctgct gcccaacgtg atcaccaagg aggtgtctca cgagatcatc 3001 aaggataggc gctttaccag cgacaagttc tttttccacg tgcctatcac actgaactat 3061 caggccgcca attccccatc taagttcaac cagagggtga atgcctacct gaaggagcac 3121 cccgagacac ctatcatcgg catcgatcgg ggcgagagaa acctgatcta tatcacagtg 3181 atcgactcca ccggcaagat cctggagcag cggagcctga acaccatcca gcagtttgat 3241 taccagaaga agctggacaa cagggagaag gagagggtgg cagcaaggca ggcctggtct 3301 gtggtgggca caatcaagga tctgaagcag ggctatctga gccaggtcat ccacgagatc 3361 gtggacctga tgatccacta ccaggccgtg gtggtgctgg agaacctgaa tttcggcttt 3421 aagagcaaga ggaccggcat cgccgagaag gccgtgtacc agcagttcga gaagatgctg 3481 atcgataagc tgaattgcct ggtgctgaag gactatccag cagagaaagt gggaggcgtg 3541 ctgaacccat accagctgac agaccagttc acctcctttg ccaagatggg cacccagtct 3601 ggcttcctgt tttacgtgcc tgccccatat acatctaaga tcgatcccct gaccggcttc 3661 gtggacccct tcgtgtggaa aaccatcaag aatcacgaga gccgcaagca cttcctggag 3721 ggcttcgact ttctgcacta cgacgtgaaa accggcgact tcatcctgca ctttaagatg 3781 aacagaaatc tgtccttcca gaggggcctg cccggcttta tgcctgcatg ggatatcgtg 3841 ttcgagaaga acgagacaca gtttgacgcc aagggcaccc ctttcatcgc cggcaagaga 3901 atcgtgccag tgatcgagaa tcacagattc accggcagat accgggacct gtatcctgcc 3961 aacgagctga tcgccctgct ggaggagaag ggcatcgtgt tcagggatgg ctccaacatc 4021 ctgccaaagc tgctggagaa tgacgattct cacgccatcg acaccatggt ggccctgatc 4081 cgcagcgtgc tgcagatgcg gaactccaat gccgccacag gcgaggacta tatcaacagc 4141 cccgtgcgcg atctgaatgg cgtgtgcttc gactcccggt ttcagaaccc agagtggccc 4201 atggacgccg atgccaatgg cgcctaccac atcgccctga agggccagct gctgctgaat 4261 cacctgaagg agagcaagga tctgaagctg cagaacggca tctccaatca ggactggctg 4321 gcctacatcc aggagctgcg caacaaaagg ccggcggcca cgaaaaaggc cggccaggca 4381 aaaaagaaaa aggaattcgg cagtggagag ggcagaggaa gtctgctaac atgcggtgac 4441 gtcgaggaga atcctggccc agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 4501 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 4561 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 4621 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 4681 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 4741 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 4801 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 4861 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 4921 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 4981 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 5041 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 5101 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 5161 gacgagctgt acaaggaatt ctaactagag ctcgctgatc agcctcgact gtgccttcta 5221 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 5281 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 5341 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata 5401 gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc 5461 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 5521 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt 5581 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta 5641 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 5701 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 5761 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 5821 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc 5881 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 5941 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata 6001 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa 6061 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg 6121 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg 6181 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 6241 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat 6301 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 6361 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 6421 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 6481 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 6541 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 6601 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 6661 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 6721 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 6781 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 6841 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 6901 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 6961 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 7021 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 7081 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 7141 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag 7201 ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 7261 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 7321 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 7381 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 7441 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 7501 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 7561 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 7621 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 7681 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 7741 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 7801 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 7861 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 7921 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 7981 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 8041 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 8101 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 8161 gtgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag 8221 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta 8281 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc 8341 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag 8401 gacgaaacac cgtaatttct actcttgtag atttccatct ctctcggtgc aggaggtttt 8461 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa 8521 gtggcaccga gtcggtgctt ttttgtttta gagctagaaa tagcaagtta aaataaggct 8581 agtccgtttt tagcgcgtgc gccaattctg cagacaaatg gctctagagg tacccgttac 8641 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc 8701 aatagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 8761 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 8821 tgacggtaaa tggcccgcct ggcattgtgc ccagtacatg accttatggg actttcctac 8881 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt 8941 ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt 9001 ttaattattt tgtgcagcga tgggggcggg ggggggggnn nnnnnnnnnn nnnnngggc //