LOCUS Exported 7578 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Level0 nSpCas9-ABE7.10 base changer, human codon optimized. ACCESSION . VERSION . KEYWORDS pFH45 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7578) AUTHORS Hahn F, Korolev A, Sanjurjo Loures L, Nekrasov V TITLE A modular cloning toolkit for genome editing in plants. JOURNAL BMC Plant Biol. 2020 Apr 23;20(1):179. doi: 10.1186/s12870-020-02388-2. PUBMED 32326901 REFERENCE 2 (bases 1 to 7578) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7578 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(25..43) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 111..215 /gene="bla" /label=AmpR promoter CDS 437..1228 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" rep_origin 1399..1987 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1888..1907 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2141..2158 /label=L4440 /note="L4440 vector, forward primer" CDS 2236..3426 /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 3427..7527 /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 7540..7560 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" ORIGIN 1 agtggctctt cagtggacga aagggcctcg tgatacgcct atttttatag gttaatgtca 61 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 121 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 181 gataaatgct tcaataatat tgaaaaagga agagtatgcg ctcacgcaac tggtccagaa 241 ccttgaccga acgcagcggt ggtaacggcg cagtggcggt tttcatggct tgttatgact 301 gttttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt 361 gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa 421 aacaaagtta aacatcatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga 481 ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg 541 ctccgcagtg gatggcggcc tgaagccaca cagcgatatt gatttgctgg ttacggtgac 601 cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc 661 ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga 721 catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa 781 tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct 841 gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga 901 tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc 961 gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta 1021 cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg 1081 cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga 1141 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccatt acgtaaaagg 1201 cgagatcacc aaggtagtcg gcaaataact gtcagaccaa gtttactcat atatacttta 1261 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 1321 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 1381 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 1441 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 1501 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc 1561 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 1621 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 1681 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 1741 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 1801 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 1861 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 1921 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 1981 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 2041 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 2101 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 2161 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatc 2221 actctgtggt ctcaaatgtc cgaagtcgag ttttcccatg agtactggat gagacacgca 2281 ttgactctcg caaagagggc ttgggatgaa cgcgaggtgc ccgtgggggc agtactcgtg 2341 cataacaatc gcgtaatcgg cgaaggttgg aataggccga tcggacgcca cgaccccact 2401 gcacatgcgg aaatcatggc ccttcgacag ggagggcttg tgatgcagaa ttatcgactt 2461 atcgatgcga cgctgtacgt cacgcttgaa ccttgcgtaa tgtgcgcggg agctatgatt 2521 cactcccgca ttggacgagt tgtattcggt gcccgcgacg ccaagacggg tgccgcaggt 2581 tcactgatgg acgtgctgca tcacccaggc atgaaccacc gggtagaaat cacagaaggc 2641 atattggcgg acgaatgtgc ggcgctgttg tccgactttt ttcgcatgcg gaggcaggag 2701 atcaaggccc agaaaaaagc acaatcctct actgactctg gtggttcttc tggtggttct 2761 agcggcagcg agactcccgg gacctcagag tccgccacac ccgaaagttc tggtggttct 2821 tctggtggtt cttccgaagt cgagttttcc catgagtact ggatgagaca cgcattgact 2881 ctcgcaaaga gggctcgaga tgaacgcgag gtgcccgtgg gggcagtact cgtgctcaac 2941 aatcgcgtaa tcggcgaagg ttggaatagg gcaatcggac tccacgaccc cactgcacat 3001 gcggaaatca tggcccttcg acagggaggg cttgtgatgc agaattatcg acttatcgat 3061 gcgacgctgt acgtcacgtt tgaaccttgc gtaatgtgcg cgggagctat gattcactcc 3121 cgcattggac gagttgtatt cggtgttcgc aacgccaaga cgggtgccgc aggttcactg 3181 atggacgtgc tgcattaccc aggcatgaac caccgggtag aaatcacaga aggcatattg 3241 gcggacgaat gtgcggcgct gttgtgttac ttttttcgca tgcccaggca ggtctttaac 3301 gcccagaaaa aagcacaatc ctctactgac tctggtggtt cttctggtgg ttctagcggc 3361 agcgagactc ccgggacctc agagtccgcc acacccgaaa gttctggtgg ttcttctggt 3421 ggttctgata aaaagtattc tattggttta gccatcggca ctaattccgt tggatgggct 3481 gtcataaccg atgaatacaa agtaccttca aagaaattta aggtgttggg gaacacagac 3541 cgtcattcga ttaaaaagaa tcttatcggt gccctcctat tcgatagtgg cgaaacggca 3601 gaggcgactc gcctgaaacg aaccgctcgg agaaggtata cacgtcgcaa gaaccgaata 3661 tgttacttac aagaaatttt tagcaatgag atggccaaag ttgacgattc tttctttcac 3721 cgtttggaag agtccttcct tgtcgaagag gacaagaaac atgaacggca ccccatcttt 3781 ggaaacatag tagatgaggt ggcatatcat gaaaagtacc caacgattta tcacctcaga 3841 aaaaagctag ttgactcaac tgataaagcg gacctgaggt taatctactt ggctcttgcc 3901 catatgataa agttccgtgg gcactttctc attgagggtg atctaaatcc ggacaactcg 3961 gatgtcgaca aactgttcat ccagttagta caaacctata atcagttgtt tgaagagaac 4021 cctataaatg caagtggcgt ggatgcgaag gctattctta gcgcccgcct ctctaaatcc 4081 cgacggctag aaaacctgat cgcacaatta cccggagaga agaaaaatgg gttgttcggt 4141 aaccttatag cgctctcact aggcctgaca ccaaatttta agtcgaactt cgacttagct 4201 gaagatgcca aattgcagct tagtaaggac acgtacgatg acgatctcga caatctactg 4261 gcacaaattg gagatcagta tgcggactta tttttggctg ccaaaaacct tagcgatgca 4321 atcctcctat ctgacatact gagagttaat actgagatta ccaaggcgcc gttatccgct 4381 tcaatgatca aaaggtacga tgaacatcac caagacttga cacttctcaa ggccctagtc 4441 cgtcagcaac tgcctgagaa atataaggaa atattctttg atcagtcgaa aaacgggtac 4501 gcaggttata ttgacggcgg agcgagtcaa gaggaattct acaagtttat caaacccata 4561 ttagagaaga tggatgggac ggaagagttg cttgtaaaac tcaatcgcga agatctactg 4621 cgaaagcagc ggactttcga caacggtagc attccacatc aaatccactt aggcgaattg 4681 catgctatac ttagaaggca ggaggatttt tatccgttcc tcaaagacaa tcgtgaaaag 4741 attgagaaaa tcctaacctt tcgcatacct tactatgtgg gacccctggc ccgagggaac 4801 tctcggttcg catggatgac aagaaagtcc gaagaaacga ttactccatg gaattttgag 4861 gaagttgtcg ataaaggtgc gtcagctcaa tcgttcatcg agaggatgac caactttgac 4921 aagaatttac cgaacgaaaa agtattgcct aagcacagtt tactttacga gtatttcaca 4981 gtgtacaatg aactcacgaa agttaagtat gtcactgagg gcatgcgtaa acccgccttt 5041 ctaagcggag aacagaagaa agcaatagta gatctgttat tcaagaccaa ccgcaaagtg 5101 acagttaagc aattgaaaga ggactacttt aagaaaattg aatgcttcga ttctgtcgag 5161 atctccgggg tagaagatcg atttaatgcg tcacttggta cgtatcatga cctcctaaag 5221 ataattaaag ataaggactt cctggataac gaagagaatg aagatatctt agaagatata 5281 gtgttgactc ttaccctctt tgaagatcgg gaaatgattg aggaaagact aaaaacatac 5341 gctcacctgt tcgacgataa ggttatgaaa cagttaaaga ggcgtcgcta tacgggctgg 5401 ggacgattgt cgcggaaact tatcaacggg ataagagaca agcaaagtgg taaaactatt 5461 ctcgattttc taaagagcga cggcttcgcc aataggaact ttatgcagct gatccatgat 5521 gactctttaa ccttcaaaga ggatatacaa aaggcacagg tttccggaca aggggactca 5581 ttgcacgaac atattgcgaa tcttgctggt tcgccagcca tcaaaaaggg catactccag 5641 acagtcaaag tagtggatga gctagttaag gtcatgggac gtcacaaacc ggaaaacatt 5701 gtaatcgaga tggcacgcga aaatcaaacg actcagaagg ggcaaaaaaa cagtcgagag 5761 cggatgaaga gaatagaaga gggtattaaa gaactgggca gccagatctt aaaggagcat 5821 cctgtggaaa atacccaatt gcagaacgag aaactttacc tctattacct acaaaatgga 5881 agggacatgt atgttgatca ggaactggac ataaaccgtt tatctgatta cgacgtcgat 5941 cacattgtac cccaatcctt tttgaaggac gattcaatcg acaataaagt gcttacacgc 6001 tcggataaga accgagggaa aagtgacaat gttccaagcg aggaagtcgt aaagaaaatg 6061 aagaactatt ggcggcagct cctaaatgcg aaactgataa cgcaaagaaa gttcgataac 6121 ttaactaaag ctgagagggg tggcttgtct gaacttgaca aggccggatt tattaaacgt 6181 cagctcgtgg aaacccgcca aatcacaaag catgttgcac agatactaga ttcccgaatg 6241 aatacgaaat acgacgagaa cgataagctg attcgggaag tcaaagtaat cactttaaag 6301 tcaaaattgg tgtcggactt cagaaaggat tttcaattct ataaagttag ggagataaat 6361 aactaccacc atgcgcacga cgcttatctt aatgccgtcg tagggaccgc actcattaag 6421 aaatacccga agctagaaag tgagtttgtg tatggtgatt acaaagttta tgacgtccgt 6481 aagatgatcg cgaaaagcga acaggagata ggcaaggcta cagccaaata cttcttttat 6541 tctaacatta tgaatttctt taagacggaa atcactctgg caaacggaga gatacgcaaa 6601 cgacctttaa ttgaaaccaa tggggagaca ggtgaaatcg tatgggataa gggccgggac 6661 ttcgcgacgg tgagaaaagt tttgtccatg ccccaagtca acatagtaaa gaaaactgag 6721 gtgcagaccg gagggttttc aaaggaatcg attcttccaa aaaggaatag tgataagctc 6781 atcgctcgta aaaaggactg ggacccgaaa aagtacggtg gcttcgatag ccctacagtt 6841 gcctattctg tcctagtagt ggcaaaagtt gagaagggaa aatccaagaa actgaagtca 6901 gtcaaagaat tattggggat aacgattatg gagcgctcgt cttttgaaaa gaaccccatc 6961 gacttccttg aggcgaaagg ttacaaggaa gtaaaaaagg atctcataat taaactacca 7021 aagtatagtc tgtttgagtt agaaaatggc cgaaaacgga tgttggctag cgccggagag 7081 cttcaaaagg ggaacgaact cgcactaccg tctaaatacg tgaatttcct gtatttagcg 7141 tcccattacg agaagttgaa aggttcacct gaagataacg aacagaagca actttttgtt 7201 gagcagcaca aacattatct cgacgaaatc atagagcaaa tttcggaatt cagtaagaga 7261 gtcatcctag ctgatgccaa tctggacaaa gtattaagcg catacaacaa gcacagggat 7321 aaacccatac gtgagcaggc ggaaaatatt atccatttgt ttactcttac caacctcggc 7381 gctccagccg cattcaagta ttttgacaca acgatagatc gcaaacgata cacttctacc 7441 aaggaggtgc tagacgcgac actgattcac caatccatca cgggattata tgaaactcgg 7501 atagatttgt cacagcttgg gggtgactct ggtggttctc ccaagaagaa gaggaaagtc 7561 taagctttga gaccacga //