LOCUS Exported 8727 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses dead Cas9 (dCas9)-ABE7.10. ACCESSION . VERSION . KEYWORDS dABE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8727) AUTHORS Smith CJ, Castanon O, Said K, Volf V, Khoshakhlagh P, Hornick A, Ferreira R, Wu CT, Guell M, Garg S, Ng AHM, Myllykallio H, Church GM TITLE Enabling large-scale genome editing at repetitive elements by reducing DNA nicking. JOURNAL Nucleic Acids Res. 2020 May 21;48(9):5183-5195. doi: 10.1093/nar/gkaa239. PUBMED 32315033 REFERENCE 2 (bases 1 to 8727) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8727 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 19..398 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 399..602 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 553..573 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 644..663 /label=T7 /note="T7 promoter, forward primer" promoter 644..662 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 676..1866 /codon_start=1 /product="adenine base editor comprising wild-type E. coli TadA fused to the evolved TadA*(7.10) mutant (Gaudelli et al., 2017)" /label=ABE(7.10) /translation="MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGW NRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVF GARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQ SSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARD EREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT FEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECA ALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGS" CDS 1867..5967 /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 5980..6000 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6009..6026 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(6049..6066) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6055..6279 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6350..6366) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6350..6366) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6363..6385) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6374..6390 /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(6398..6428) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6443..6464 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6581..6598) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6752..7340) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6832..6851) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7511..8371) /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 8134..8153 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8372..8476) /gene="bla" /label=AmpR promoter primer_bind complement(8555..8574) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg 61 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc 121 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 181 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 241 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 301 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 361 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca 421 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 481 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 541 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 601 ctggtttagt gaaccgtcag atccgctaga gatccgcggc cgctaatacg actcactata 661 gggagagccg ccaccatgtc cgaagtcgag ttttcccatg agtactggat gagacacgca 721 ttgactctcg caaagagggc ttgggatgaa cgcgaggtgc ccgtgggggc agtactcgtg 781 cataacaatc gcgtaatcgg cgaaggttgg aataggccga tcggacgcca cgaccccact 841 gcacatgcgg aaatcatggc ccttcgacag ggagggcttg tgatgcagaa ttatcgactt 901 atcgatgcga cgctgtacgt cacgcttgaa ccttgcgtaa tgtgcgcggg agctatgatt 961 cactcccgca ttggacgagt tgtattcggt gcccgcgacg ccaagacggg tgccgcaggt 1021 tcactgatgg acgtgctgca tcacccaggc atgaaccacc gggtagaaat cacagaaggc 1081 atattggcgg acgaatgtgc ggcgctgttg tccgactttt ttcgcatgcg gaggcaggag 1141 atcaaggccc agaaaaaagc acaatcctct actgactctg gtggttcttc tggtggttct 1201 agcggcagcg agactcccgg gacctcagag tccgccacac ccgaaagttc tggtggttct 1261 tctggtggtt cttccgaagt cgagttttcc catgagtact ggatgagaca cgcattgact 1321 ctcgcaaaga gggctcgaga tgaacgcgag gtgcccgtgg gggcagtact cgtgctcaac 1381 aatcgcgtaa tcggcgaagg ttggaatagg gcaatcggac tccacgaccc cactgcacat 1441 gcggaaatca tggcccttcg acagggaggg cttgtgatgc agaattatcg acttatcgat 1501 gcgacgctgt acgtcacgtt tgaaccttgc gtaatgtgcg cgggagctat gattcactcc 1561 cgcattggac gagttgtatt cggtgttcgc aacgccaaga cgggtgccgc aggttcactg 1621 atggacgtgc tgcattaccc aggcatgaac caccgggtag aaatcacaga aggcatattg 1681 gcggacgaat gtgcggcgct gttgtgttac ttttttcgca tgcccaggca ggtctttaac 1741 gcccagaaaa aagcacaatc ctctactgac tctggtggtt cttctggtgg ttctagcggc 1801 agcgagactc ccgggacctc agagtccgcc acacccgaaa gttctggtgg ttcttctggt 1861 ggttctgata aaaagtattc tattggttta gccatcggca ctaattccgt tggatgggct 1921 gtcataaccg atgaatacaa agtaccttca aagaaattta aggtgttggg gaacacagac 1981 cgtcattcga ttaaaaagaa tcttatcggt gccctcctat tcgatagtgg cgaaacggca 2041 gaggcgactc gcctgaaacg aaccgctcgg agaaggtata cacgtcgcaa gaaccgaata 2101 tgttacttac aagaaatttt tagcaatgag atggccaaag ttgacgattc tttctttcac 2161 cgtttggaag agtccttcct tgtcgaagag gacaagaaac atgaacggca ccccatcttt 2221 ggaaacatag tagatgaggt ggcatatcat gaaaagtacc caacgattta tcacctcaga 2281 aaaaagctag ttgactcaac tgataaagcg gacctgaggt taatctactt ggctcttgcc 2341 catatgataa agttccgtgg gcactttctc attgagggtg atctaaatcc ggacaactcg 2401 gatgtcgaca aactgttcat ccagttagta caaacctata atcagttgtt tgaagagaac 2461 cctataaatg caagtggcgt ggatgcgaag gctattctta gcgcccgcct ctctaaatcc 2521 cgacggctag aaaacctgat cgcacaatta cccggagaga agaaaaatgg gttgttcggt 2581 aaccttatag cgctctcact aggcctgaca ccaaatttta agtcgaactt cgacttagct 2641 gaagatgcca aattgcagct tagtaaggac acgtacgatg acgatctcga caatctactg 2701 gcacaaattg gagatcagta tgcggactta tttttggctg ccaaaaacct tagcgatgca 2761 atcctcctat ctgacatact gagagttaat actgagatta ccaaggcgcc gttatccgct 2821 tcaatgatca aaaggtacga tgaacatcac caagacttga cacttctcaa ggccctagtc 2881 cgtcagcaac tgcctgagaa atataaggaa atattctttg atcagtcgaa aaacgggtac 2941 gcaggttata ttgacggcgg agcgagtcaa gaggaattct acaagtttat caaacccata 3001 ttagagaaga tggatgggac ggaagagttg cttgtaaaac tcaatcgcga agatctactg 3061 cgaaagcagc ggactttcga caacggtagc attccacatc aaatccactt aggcgaattg 3121 catgctatac ttagaaggca ggaggatttt tatccgttcc tcaaagacaa tcgtgaaaag 3181 attgagaaaa tcctaacctt tcgcatacct tactatgtgg gacccctggc ccgagggaac 3241 tctcggttcg catggatgac aagaaagtcc gaagaaacga ttactccatg gaattttgag 3301 gaagttgtcg ataaaggtgc gtcagctcaa tcgttcatcg agaggatgac caactttgac 3361 aagaatttac cgaacgaaaa agtattgcct aagcacagtt tactttacga gtatttcaca 3421 gtgtacaatg aactcacgaa agttaagtat gtcactgagg gcatgcgtaa acccgccttt 3481 ctaagcggag aacagaagaa agcaatagta gatctgttat tcaagaccaa ccgcaaagtg 3541 acagttaagc aattgaaaga ggactacttt aagaaaattg aatgcttcga ttctgtcgag 3601 atctccgggg tagaagatcg atttaatgcg tcacttggta cgtatcatga cctcctaaag 3661 ataattaaag ataaggactt cctggataac gaagagaatg aagatatctt agaagatata 3721 gtgttgactc ttaccctctt tgaagatcgg gaaatgattg aggaaagact aaaaacatac 3781 gctcacctgt tcgacgataa ggttatgaaa cagttaaaga ggcgtcgcta tacgggctgg 3841 ggacgattgt cgcggaaact tatcaacggg ataagagaca agcaaagtgg taaaactatt 3901 ctcgattttc taaagagcga cggcttcgcc aataggaact ttatgcagct gatccatgat 3961 gactctttaa ccttcaaaga ggatatacaa aaggcacagg tttccggaca aggggactca 4021 ttgcacgaac atattgcgaa tcttgctggt tcgccagcca tcaaaaaggg catactccag 4081 acagtcaaag tagtggatga gctagttaag gtcatgggac gtcacaaacc ggaaaacatt 4141 gtaatcgaga tggcacgcga aaatcaaacg actcagaagg ggcaaaaaaa cagtcgagag 4201 cggatgaaga gaatagaaga gggtattaaa gaactgggca gccagatctt aaaggagcat 4261 cctgtggaaa atacccaatt gcagaacgag aaactttacc tctattacct acaaaatgga 4321 agggacatgt atgttgatca ggaactggac ataaaccgtt tatctgatta cgacgtcgat 4381 gccattgtac cccaatcctt tttgaaggac gattcaatcg acaataaagt gcttacacgc 4441 tcggataaga accgagggaa aagtgacaat gttccaagcg aggaagtcgt aaagaaaatg 4501 aagaactatt ggcggcagct cctaaatgcg aaactgataa cgcaaagaaa gttcgataac 4561 ttaactaaag ctgagagggg tggcttgtct gaacttgaca aggccggatt tattaaacgt 4621 cagctcgtgg aaacccgcca aatcacaaag catgttgcac agatactaga ttcccgaatg 4681 aatacgaaat acgacgagaa cgataagctg attcgggaag tcaaagtaat cactttaaag 4741 tcaaaattgg tgtcggactt cagaaaggat tttcaattct ataaagttag ggagataaat 4801 aactaccacc atgcgcacga cgcttatctt aatgccgtcg tagggaccgc actcattaag 4861 aaatacccga agctagaaag tgagtttgtg tatggtgatt acaaagttta tgacgtccgt 4921 aagatgatcg cgaaaagcga acaggagata ggcaaggcta cagccaaata cttcttttat 4981 tctaacatta tgaatttctt taagacggaa atcactctgg caaacggaga gatacgcaaa 5041 cgacctttaa ttgaaaccaa tggggagaca ggtgaaatcg tatgggataa gggccgggac 5101 ttcgcgacgg tgagaaaagt tttgtccatg ccccaagtca acatagtaaa gaaaactgag 5161 gtgcagaccg gagggttttc aaaggaatcg attcttccaa aaaggaatag tgataagctc 5221 atcgctcgta aaaaggactg ggacccgaaa aagtacggtg gcttcgatag ccctacagtt 5281 gcctattctg tcctagtagt ggcaaaagtt gagaagggaa aatccaagaa actgaagtca 5341 gtcaaagaat tattggggat aacgattatg gagcgctcgt cttttgaaaa gaaccccatc 5401 gacttccttg aggcgaaagg ttacaaggaa gtaaaaaagg atctcataat taaactacca 5461 aagtatagtc tgtttgagtt agaaaatggc cgaaaacgga tgttggctag cgccggagag 5521 cttcaaaagg ggaacgaact cgcactaccg tctaaatacg tgaatttcct gtatttagcg 5581 tcccattacg agaagttgaa aggttcacct gaagataacg aacagaagca actttttgtt 5641 gagcagcaca aacattatct cgacgaaatc atagagcaaa tttcggaatt cagtaagaga 5701 gtcatcctag ctgatgccaa tctggacaaa gtattaagcg catacaacaa gcacagggat 5761 aaacccatac gtgagcaggc ggaaaatatt atccatttgt ttactcttac caacctcggc 5821 gctccagccg cattcaagta ttttgacaca acgatagatc gcaaacgata cacttctacc 5881 aaggaggtgc tagacgcgac actgattcac caatccatca cgggattata tgaaactcgg 5941 atagatttgt cacagcttgg gggtgactct ggtggttctc ccaagaagaa gaggaaagtc 6001 taaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc 6061 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 6121 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 6181 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 6241 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 6301 gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt 6361 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 6421 agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 6481 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 6541 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 6601 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 6661 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 6721 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 6781 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 6841 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 6901 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 6961 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 7021 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 7081 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 7141 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 7201 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 7261 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 7321 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 7381 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 7441 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 7501 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 7561 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 7621 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 7681 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 7741 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 7801 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 7861 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 7921 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 7981 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 8041 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 8101 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 8161 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 8221 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 8281 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 8341 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 8401 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 8461 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag 8521 atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt 8581 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa 8641 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta 8701 ggcgttttgc gctgcttcgc gatgtac //