LOCUS Exported 10754 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION bacterial expression of AsCpf1-2NLS. ACCESSION . VERSION . KEYWORDS AsCpf1-2NLS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10754) AUTHORS Moreno-Mateos MA, Fernandez JP, Rouet R, Vejnar CE, Lane MA, Mis E, Khokha MK, Doudna JA, Giraldez AJ TITLE CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. JOURNAL Nat Commun. 2017 Dec 8;8(1):2024. doi: 10.1038/s41467-017-01836-2. PUBMED 29222508 REFERENCE 2 (bases 1 to 10754) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10754 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(18..36) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 104..208 /gene="bla" /label=AmpR promoter CDS 209..1069 /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(427..446) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1240..1828 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1729..1748 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1982..1999 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2014..2156 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2082..2101) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2242..2264 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2258..2449) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind 3907..3926 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4010..4029 /label=T7 /note="T7 promoter, forward primer" promoter 4010..4028 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 4117..4139 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 4159..4176 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 4186..5286 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" primer_bind 5260..5283 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 5341..5361 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 5371..9291 /codon_start=1 /product="CRISPR-associated protein from Acidaminococcus sp. BV3L6 (Zetsche et al., 2015)" /label=AsCpf1 /translation="MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRN AIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFD KFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHF ENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL RNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYK ALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEP LEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSID LSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLK DQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFF HVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYD VKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEN HRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQM RNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKES KDLKLQNGISNQDWLAYIQELRN" CDS 9295..9321 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 9340..9360 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9367..9387 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9427..10131 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(9569..9587) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 9837..9856 /label=mCherry-F /note="mCherry, forward primer" CDS 10138..10158 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(10290..10308) /label=T7 Term /note="T7 terminator, reverse primer" terminator 10304..10351 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(10717..10745) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" ORIGIN 1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt 3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc 3721 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc 3781 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc 3841 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc 3901 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc 3961 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc 4021 actataggga gaccacaacg gtttccctct agtgccggct ccggagagct ctttaattaa 4081 gcggccgccc tgcaggactc gagttctaga aataattttg tttaacttta agaaggagat 4141 atacatatga aatcttctca ccatcaccat caccatggtt cttctatgaa aatcgaagaa 4201 ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt 4261 aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa 4321 gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac 4381 gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg 4441 ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt 4501 gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac 4561 ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag 4621 agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac 4681 gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat 4741 aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg 4801 aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg 4861 accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta 4921 acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca 4981 ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg 5041 ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg 5101 aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc 5161 cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt 5221 actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg 5281 cagactaatt cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag 5341 gaaaacctgt acttccaatc caatgccacc atgacccagt tcgaggggtt taccaatctg 5401 tatcaagtga gcaagacgct gcgctttgaa ctgatcccac agggaaaaac cttaaaacat 5461 attcaagagc agggctttat cgaagaagat aaggcccgta atgaccatta caaagagtta 5521 aagccgatta ttgatcgtat ctacaagacc tatgcggacc agtgcttaca attggtacag 5581 cttgattggg agaacctctc tgccgccatc gattcctatc gtaaagaaaa aactgaagaa 5641 acgcgcaacg ccctgattga agagcaggcc acctatcgta acgcgattca tgactatttt 5701 attggccgta cggacaatct gacggacgcg atcaacaagc gccatgcgga gatttacaaa 5761 ggactgttta aggctgaact gttcaatggt aaggtcctta aacagcttgg gaccgtcaca 5821 acgacggaac atgaaaacgc gttattacgt agcttcgaca agtttaccac gtatttctcc 5881 ggcttttacg aaaatcgcaa aaacgttttc agtgccgagg atatttccac tgctatccct 5941 catcgcattg tgcaagacaa cttcccaaaa ttcaaagaaa attgtcatat cttcacccgc 6001 ttaatcaccg ctgtaccgtc cctgcgtgag catttcgaaa acgtgaaaaa ggccattggt 6061 atcttcgtgt ctacttcgat tgaggaggta ttttcctttc cattctataa tcagctgctg 6121 acccagaccc aaattgatct gtacaaccag ctgcttggcg gtatttctcg tgaagcagga 6181 accgaaaaaa tcaaagggtt gaacgaggtg cttaatctgg caatccagaa aaatgatgaa 6241 accgcccaca tcattgcttc gttacctcat cgttttatcc cgttgttcaa gcaaatttta 6301 agtgatcgca atacgctgtc gtttattctg gaagaattca aaagtgatga agaggtaatt 6361 cagtcgtttt gcaaatataa aaccctgtta cgtaacgaaa atgtcctgga aacagccgag 6421 gctttgttta acgaactgaa tagcattgac ctgacgcata tctttattag ccacaaaaaa 6481 ttagagacca tctcatcagc tctgtgcgat cattgggata cactgcgcaa tgcgctgtat 6541 gaacgtcgta tttcggaatt gactggcaaa atcactaaaa gcgcgaaaga gaaagtacag 6601 cgctcgctta aacatgaaga tatcaacctg caggagatca tcagcgccgc gggtaaagaa 6661 ctgtcggagg catttaaaca gaagacgagc gagattctgt cccacgcaca tgccgcctta 6721 gaccagccgc tcccgaccac tctgaagaaa caggaagaga aagaaatcct taaaagtcaa 6781 ctggacagtt tactgggtct ctatcatctg ctggattggt ttgcggtaga cgaaagcaat 6841 gaagtggatc cggagtttag tgcccgtctg acaggaatca agctggaaat ggagccttcg 6901 cttagcttct acaacaaagc ccgcaattat gccacgaaaa aaccctatag tgtcgaaaaa 6961 tttaaactca actttcaaat gccgaccctt gcgtcgggct gggatgtcaa caaagaaaaa 7021 aacaacggag ctattctgtt cgttaaaaat ggtctgtact acctgggcat catgccgaaa 7081 cagaaaggtc gctacaaagc cctttcgttc gagcccacgg aaaaaacaag cgaaggcttc 7141 gacaaaatgt actacgatta ctttccggat gcagcaaaaa tgatcccgaa atgttccaca 7201 cagctgaaag ccgttacagc acattttcag acgcacacca cccccatctt actgtccaac 7261 aattttattg aaccgctgga gattactaaa gaaatttatg atttgaacaa tccggaaaaa 7321 gagccaaaaa agtttcaaac cgcctacgct aaaaaaaccg gggatcagaa agggtaccgc 7381 gaagcgttgt gcaagtggat tgatttcacc cgcgattttc tcagtaaata taccaagact 7441 acctcgattg acctgagctc actgcgcccg agctctcaat ataaggattt gggtgagtac 7501 tatgctgaat taaacccttt attgtaccac atttcttttc agcgcatcgc cgaaaaggaa 7561 attatggacg cagtcgaaac cgggaaactg tacctgttcc agatctataa taaggacttc 7621 gccaaaggac atcatggcaa accgaacctg cacacccttt actggaccgg gcttttctct 7681 ccggaaaatt tggcgaaaac ctcgatcaag cttaacggtc aagctgagct gttttaccgt 7741 ccaaaatccc gcatgaagcg catggcgcat cgtttaggtg aaaaaatgct gaataagaaa 7801 ctgaaagatc agaaaacccc tatcccggat accctctacc aggaactgta tgattacgtg 7861 aaccatcgtc tctcgcatga cctgtcagac gaagcgcgtg cgttactgcc caatgtaatc 7921 acaaaagaag tttcgcatga aattattaaa gatcgtcgtt ttacatctga taaattcttt 7981 tttcatgttc cgatcaccct caactatcag gccgcaaaca gtccaagtaa gtttaaccag 8041 cgcgttaatg cttacctgaa ggaacatccg gagactccga ttattggaat tgatcgcggt 8101 gaacgtaatt tgatctatat cactgtgatc gatagtaccg gtaagattct ggagcagcgc 8161 agcttgaaca caattcaaca gtttgattat cagaaaaaat tagacaaccg cgaaaaagag 8221 cgcgtggctg cccgtcaggc gtggtctgtt gtcggtacca ttaaagatct gaagcagggc 8281 tatctttctc aggttattca cgaaattgta gatctgatga tccattatca ggcggttgtt 8341 gtgttggaga atctcaattt cggttttaag agtaagcgca caggcatcgc tgaaaaagca 8401 gtttatcagc agtttgaaaa aatgctgatc gacaaattga actgtttagt tctcaaagat 8461 tacccagcgg aaaaggtggg cggagtgctg aatccgtacc aattaacgga tcaattcact 8521 tccttcgcaa agatgggtac ccaaagcggc tttctgttct atgtgccggc cccgtatacc 8581 tcgaaaatcg atccactgac gggcttcgta gatccgttcg tgtggaaaac cattaaaaat 8641 catgaaagtc gtaaacattt tctcgaaggc ttcgacttcc tgcactacga cgtgaaaact 8701 ggcgatttca ttctgcattt taaaatgaac cgcaaccttt cgtttcagcg cggtctgccg 8761 ggctttatgc cggcttggga cattgttttt gagaaaaatg aaacccagtt tgatgctaaa 8821 ggcactcctt tcatcgccgg taaacgcatc gtacctgtga ttgaaaacca tcgttttaca 8881 gggcgttacc gtgatttata cccggcgaac gaattgatcg cgctgctgga ggaaaagggc 8941 atcgttttcc gtgacggctc caatattctg ccgaaattac tggaaaacga cgattcacac 9001 gcaattgata ccatggtcgc actgattcgc tcagtcttac agatgcgtaa ctctaatgca 9061 gccacaggag aagattatat taattcgcca gtccgcgatt tgaacggtgt ttgcttcgac 9121 agccgttttc agaatcctga atggccgatg gacgctgatg ccaacggagc ttatcatatc 9181 gccctgaaag gccagctcct gctgaaccac ctgaaggaaa gcaaagatct gaaattgcag 9241 aacggcatta gcaaccagga ctggttagca tacatccagg aactgcgtaa cgcctatccc 9301 tatgacgtgc ccgattatgc cagcctgggc agcggctccc ccaagaaaaa acgcaaggtg 9361 gaagatccta agaaaaagcg gaaagtggac taaattggta gtgggagcaa cggcagcagc 9421 ggatccgtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc 9481 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag 9541 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg 9601 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag 9661 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag 9721 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctccag 9781 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc 9841 gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac 9901 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac 9961 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac 10021 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac 10081 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa ggggtcacct 10141 aagaaaaaac gaaaagttga gtaataacat tggaagtgga taacggatcc gcgatcgcgg 10201 cgcgccacct ggtggccggc cggtaccacg cgtgcgcgct gatccggctg ctaacaaagc 10261 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg 10321 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc 10381 acaggacggg tgtggtcgcc atgatcgcgt agtcgatagt ggctccaagt agcgaagcga 10441 gcaggactgg gcggcggcca aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa 10501 attgcatcaa cgcatatagc gctagcagca cgccatagtg actggcgatg ctgtcggaat 10561 ggacgatatc ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat 10621 ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct 10681 gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct 10741 gtcaaacatg agaa //