LOCUS Exported 8454 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses hA3H-BE3 in mammalian cells. ACCESSION . VERSION . KEYWORDS pCMV-hA3H-BE3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8454) AUTHORS Wang X, Li J, Wang Y, Yang V, Wei J, Wu J, Wang R, Huang X, Chen J, Yang L TITLE Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. JOURNAL Nat Biotechnol., 2018 REFERENCE 2 (bases 1 to 8454) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8454 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 137..516 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 517..720 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 671..691 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 762..781 /label=T7 /note="T7 promoter, forward primer" promoter 762..780 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 788..797 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1451..5551 /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 5564..5812 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5825..5845 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5854..5871 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5894..5911) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5900..6124 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6195..6211) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6195..6211) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6208..6230) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6219..6235 /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(6243..6273) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6288..6309 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6426..6443) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6597..7185) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6677..6696) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7356..8216) /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 7979..7998 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8217..8321) /gene="bla" /label=AmpR promoter primer_bind complement(8400..8419) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc 61 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg 121 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg 181 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 241 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 301 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 361 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 421 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 481 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 541 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 601 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 661 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 721 ggtttagtga accgtcagat ccgctagaga tccgcggccg ctaatacgac tcactatagg 781 gagagccgcc accatggctc tgttaacagc cgaaacattc cgcttacagt ttaacaacaa 841 gcgccgcctc agaaggcctt actacccgag gaaggccctc ttgtgttacc agctgacgcc 901 gcagaatggc tccacgccca cgagaggcta ctttgaaaac aagaaaaagt gccatgcaga 961 aatttgcttt attaacgaga tcaagtccat gggactggac gaaacgcagt gctaccaagt 1021 cacctgttac ctcacgtgga gcccctgctc ctcctgtgcc tgggagctgg ttgacttcat 1081 caaggctcac gaccatctga acctgggcat cttcgcctcc cgcctgtact accactggtg 1141 caagccccag cagaaggggc tgcggcttct gtgtggatcc caggtcccgg tggaggtcat 1201 gggcttccca gagtttgctg actgctggga aaactttgtg gaccacgaga aaccgctttc 1261 cttcaacccc tataagatgt tagaggagct agataaaaac agtcgagcca taaagcgacg 1321 gcttgagagg ataaaggggc tccttcttgt tcttttagat tccaggggta cgtgcgcagg 1381 gtcgttacat ggatatattg tgagcggcag cgagactccc gggacctcag agtccgccac 1441 acccgaaagt gataaaaagt attctattgg tttagccatc ggcactaatt ccgttggatg 1501 ggctgtcata accgatgaat acaaagtacc ttcaaagaaa tttaaggtgt tggggaacac 1561 agaccgtcat tcgattaaaa agaatcttat cggtgccctc ctattcgata gtggcgaaac 1621 ggcagaggcg actcgcctga aacgaaccgc tcggagaagg tatacacgtc gcaagaaccg 1681 aatatgttac ttacaagaaa tttttagcaa tgagatggcc aaagttgacg attctttctt 1741 tcaccgtttg gaagagtcct tccttgtcga agaggacaag aaacatgaac ggcaccccat 1801 ctttggaaac atagtagatg aggtggcata tcatgaaaag tacccaacga tttatcacct 1861 cagaaaaaag ctagttgact caactgataa agcggacctg aggttaatct acttggctct 1921 tgcccatatg ataaagttcc gtgggcactt tctcattgag ggtgatctaa atccggacaa 1981 ctcggatgtc gacaaactgt tcatccagtt agtacaaacc tataatcagt tgtttgaaga 2041 gaaccctata aatgcaagtg gcgtggatgc gaaggctatt cttagcgccc gcctctctaa 2101 atcccgacgg ctagaaaacc tgatcgcaca attacccgga gagaagaaaa atgggttgtt 2161 cggtaacctt atagcgctct cactaggcct gacaccaaat tttaagtcga acttcgactt 2221 agctgaagat gccaaattgc agcttagtaa ggacacgtac gatgacgatc tcgacaatct 2281 actggcacaa attggagatc agtatgcgga cttatttttg gctgccaaaa accttagcga 2341 tgcaatcctc ctatctgaca tactgagagt taatactgag attaccaagg cgccgttatc 2401 cgcttcaatg atcaaaaggt acgatgaaca tcaccaagac ttgacacttc tcaaggccct 2461 agtccgtcag caactgcctg agaaatataa ggaaatattc tttgatcagt cgaaaaacgg 2521 gtacgcaggt tatattgacg gcggagcgag tcaagaggaa ttctacaagt ttatcaaacc 2581 catattagag aagatggatg ggacggaaga gttgcttgta aaactcaatc gcgaagatct 2641 actgcgaaag cagcggactt tcgacaacgg tagcattcca catcaaatcc acttaggcga 2701 attgcatgct atacttagaa ggcaggagga tttttatccg ttcctcaaag acaatcgtga 2761 aaagattgag aaaatcctaa cctttcgcat accttactat gtgggacccc tggcccgagg 2821 gaactctcgg ttcgcatgga tgacaagaaa gtccgaagaa acgattactc catggaattt 2881 tgaggaagtt gtcgataaag gtgcgtcagc tcaatcgttc atcgagagga tgaccaactt 2941 tgacaagaat ttaccgaacg aaaaagtatt gcctaagcac agtttacttt acgagtattt 3001 cacagtgtac aatgaactca cgaaagttaa gtatgtcact gagggcatgc gtaaacccgc 3061 ctttctaagc ggagaacaga agaaagcaat agtagatctg ttattcaaga ccaaccgcaa 3121 agtgacagtt aagcaattga aagaggacta ctttaagaaa attgaatgct tcgattctgt 3181 cgagatctcc ggggtagaag atcgatttaa tgcgtcactt ggtacgtatc atgacctcct 3241 aaagataatt aaagataagg acttcctgga taacgaagag aatgaagata tcttagaaga 3301 tatagtgttg actcttaccc tctttgaaga tcgggaaatg attgaggaaa gactaaaaac 3361 atacgctcac ctgttcgacg ataaggttat gaaacagtta aagaggcgtc gctatacggg 3421 ctggggacga ttgtcgcgga aacttatcaa cgggataaga gacaagcaaa gtggtaaaac 3481 tattctcgat tttctaaaga gcgacggctt cgccaatagg aactttatgc agctgatcca 3541 tgatgactct ttaaccttca aagaggatat acaaaaggca caggtttccg gacaagggga 3601 ctcattgcac gaacatattg cgaatcttgc tggttcgcca gccatcaaaa agggcatact 3661 ccagacagtc aaagtagtgg atgagctagt taaggtcatg ggacgtcaca aaccggaaaa 3721 cattgtaatc gagatggcac gcgaaaatca aacgactcag aaggggcaaa aaaacagtcg 3781 agagcggatg aagagaatag aagagggtat taaagaactg ggcagccaga tcttaaagga 3841 gcatcctgtg gaaaataccc aattgcagaa cgagaaactt tacctctatt acctacaaaa 3901 tggaagggac atgtatgttg atcaggaact ggacataaac cgtttatctg attacgacgt 3961 cgatcacatt gtaccccaat cctttttgaa ggacgattca atcgacaata aagtgcttac 4021 acgctcggat aagaaccgag ggaaaagtga caatgttcca agcgaggaag tcgtaaagaa 4081 aatgaagaac tattggcggc agctcctaaa tgcgaaactg ataacgcaaa gaaagttcga 4141 taacttaact aaagctgaga ggggtggctt gtctgaactt gacaaggccg gatttattaa 4201 acgtcagctc gtggaaaccc gccaaatcac aaagcatgtt gcacagatac tagattcccg 4261 aatgaatacg aaatacgacg agaacgataa gctgattcgg gaagtcaaag taatcacttt 4321 aaagtcaaaa ttggtgtcgg acttcagaaa ggattttcaa ttctataaag ttagggagat 4381 aaataactac caccatgcgc acgacgctta tcttaatgcc gtcgtaggga ccgcactcat 4441 taagaaatac ccgaagctag aaagtgagtt tgtgtatggt gattacaaag tttatgacgt 4501 ccgtaagatg atcgcgaaaa gcgaacagga gataggcaag gctacagcca aatacttctt 4561 ttattctaac attatgaatt tctttaagac ggaaatcact ctggcaaacg gagagatacg 4621 caaacgacct ttaattgaaa ccaatgggga gacaggtgaa atcgtatggg ataagggccg 4681 ggacttcgcg acggtgagaa aagttttgtc catgccccaa gtcaacatag taaagaaaac 4741 tgaggtgcag accggagggt tttcaaagga atcgattctt ccaaaaagga atagtgataa 4801 gctcatcgct cgtaaaaagg actgggaccc gaaaaagtac ggtggcttcg atagccctac 4861 agttgcctat tctgtcctag tagtggcaaa agttgagaag ggaaaatcca agaaactgaa 4921 gtcagtcaaa gaattattgg ggataacgat tatggagcgc tcgtcttttg aaaagaaccc 4981 catcgacttc cttgaggcga aaggttacaa ggaagtaaaa aaggatctca taattaaact 5041 accaaagtat agtctgtttg agttagaaaa tggccgaaaa cggatgttgg ctagcgccgg 5101 agagcttcaa aaggggaacg aactcgcact accgtctaaa tacgtgaatt tcctgtattt 5161 agcgtcccat tacgagaagt tgaaaggttc acctgaagat aacgaacaga agcaactttt 5221 tgttgagcag cacaaacatt atctcgacga aatcatagag caaatttcgg aattcagtaa 5281 gagagtcatc ctagctgatg ccaatctgga caaagtatta agcgcataca acaagcacag 5341 ggataaaccc atacgtgagc aggcggaaaa tattatccat ttgtttactc ttaccaacct 5401 cggcgctcca gccgcattca agtattttga cacaacgata gatcgcaaac gatacacttc 5461 taccaaggag gtgctagacg cgacactgat tcaccaatcc atcacgggat tatatgaaac 5521 tcggatagat ttgtcacagc ttgggggtga ctctggtggt tctactaatc tgtcagatat 5581 tattgaaaag gagaccggta agcaactggt tatccaggaa tccatcctca tgctcccaga 5641 ggaggtggaa gaagtcattg ggaacaagcc ggaaagcgat atactcgtgc acaccgccta 5701 cgacgagagc accgacgaga atgtcatgct tctgactagc gacgcccctg aatacaagcc 5761 ttgggctctg gtcatacagg atagcaacgg tgagaacaag attaagatgc tctctggtgg 5821 ttctcccaag aagaagagga aagtctaacc ggtcatcatc accatcacca ttgagtttaa 5881 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 5941 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 6001 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 6061 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct 6121 atggcttctg aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc 6181 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 6241 cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa 6301 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 6361 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 6421 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 6481 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 6541 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 6601 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 6661 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 6721 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 6781 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 6841 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 6901 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 6961 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 7021 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 7081 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 7141 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 7201 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 7261 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 7321 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 7381 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 7441 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 7501 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 7561 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 7621 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 7681 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 7741 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 7801 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 7861 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 7921 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 7981 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 8041 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 8101 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 8161 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 8221 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 8281 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 8341 ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc gactctcagt 8401 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtg //