LOCUS Exported 8472 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses cAICDA-BE3 in mammalian cells. ACCESSION . VERSION . KEYWORDS pCMV-cAICDA-BE3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8472) 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 8472) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8472 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(10..29) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 201..580 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 581..784 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 735..755 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 826..845 /label=T7 /note="T7 promoter, forward primer" promoter 826..844 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1533..5633 /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 5646..5894 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5907..5927 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5936..5953 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5976..5993) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5982..6206 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6277..6293) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6277..6293) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6290..6312) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6301..6317 /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(6325..6355) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6370..6391 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6508..6525) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6679..7267) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6759..6778) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7438..8298) /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 8061..8080 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8299..8403) /gene="bla" /label=AmpR promoter ORIGIN 1 gactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg 61 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt 121 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt 181 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac 241 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 301 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 361 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 421 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 481 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 541 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 601 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 661 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 721 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 781 agctggttta gtgaaccgtc agatccgcta gagatccgcg gccgctaata cgactcacta 841 tagggagagc cgccaccatg agcaagctgg acagtgtgct gctgactcag aggaagttta 901 tttaccacta taagaatgtg cgctgggctc gtgggaggaa cgagacctac ctctgttttg 961 tggtcaagaa acgcaacagt cccgactcgc tctccttcga cttcggacac ctgcgcaatc 1021 gttctggctg ccatgtggag cttctcttcc tgagctatct tggggtactg tgcccaggtt 1081 tcttgggttc cggtgtggat ggtgtcaggg tggcttatgc catcacctgg ttctgttcct 1141 ggtcaccctg ttcaaactgt gcccatcgcc tttctcgctt catgtctcag atgcccaacc 1201 tgcggctgcg catcttcgtc tcgcgcctct acttctgtga cgaggaggac agtcaagaga 1261 gagagggact ccgttgcttg cagagggcag gtgtgcaagt gacagtcatg acctataaag 1321 attttttcta ctgttggcaa acctttgtgg ctcaaaatca gaaggctttc aaggcttggg 1381 acgaccttca ccagaactct atccgactgt ctcggaaact acagcgaatc ctgcagccta 1441 gtgagtctga agacctgagg gatggcttcg ctctgctggg ccttagcggc agcgagactc 1501 ccgggacctc agagtccgcc acacccgaaa gtgataaaaa gtattctatt ggtttagcca 1561 tcggcactaa ttccgttgga tgggctgtca taaccgatga atacaaagta ccttcaaaga 1621 aatttaaggt gttggggaac acagaccgtc attcgattaa aaagaatctt atcggtgccc 1681 tcctattcga tagtggcgaa acggcagagg cgactcgcct gaaacgaacc gctcggagaa 1741 ggtatacacg tcgcaagaac cgaatatgtt acttacaaga aatttttagc aatgagatgg 1801 ccaaagttga cgattctttc tttcaccgtt tggaagagtc cttccttgtc gaagaggaca 1861 agaaacatga acggcacccc atctttggaa acatagtaga tgaggtggca tatcatgaaa 1921 agtacccaac gatttatcac ctcagaaaaa agctagttga ctcaactgat aaagcggacc 1981 tgaggttaat ctacttggct cttgcccata tgataaagtt ccgtgggcac tttctcattg 2041 agggtgatct aaatccggac aactcggatg tcgacaaact gttcatccag ttagtacaaa 2101 cctataatca gttgtttgaa gagaacccta taaatgcaag tggcgtggat gcgaaggcta 2161 ttcttagcgc ccgcctctct aaatcccgac ggctagaaaa cctgatcgca caattacccg 2221 gagagaagaa aaatgggttg ttcggtaacc ttatagcgct ctcactaggc ctgacaccaa 2281 attttaagtc gaacttcgac ttagctgaag atgccaaatt gcagcttagt aaggacacgt 2341 acgatgacga tctcgacaat ctactggcac aaattggaga tcagtatgcg gacttatttt 2401 tggctgccaa aaaccttagc gatgcaatcc tcctatctga catactgaga gttaatactg 2461 agattaccaa ggcgccgtta tccgcttcaa tgatcaaaag gtacgatgaa catcaccaag 2521 acttgacact tctcaaggcc ctagtccgtc agcaactgcc tgagaaatat aaggaaatat 2581 tctttgatca gtcgaaaaac gggtacgcag gttatattga cggcggagcg agtcaagagg 2641 aattctacaa gtttatcaaa cccatattag agaagatgga tgggacggaa gagttgcttg 2701 taaaactcaa tcgcgaagat ctactgcgaa agcagcggac tttcgacaac ggtagcattc 2761 cacatcaaat ccacttaggc gaattgcatg ctatacttag aaggcaggag gatttttatc 2821 cgttcctcaa agacaatcgt gaaaagattg agaaaatcct aacctttcgc ataccttact 2881 atgtgggacc cctggcccga gggaactctc ggttcgcatg gatgacaaga aagtccgaag 2941 aaacgattac tccatggaat tttgaggaag ttgtcgataa aggtgcgtca gctcaatcgt 3001 tcatcgagag gatgaccaac tttgacaaga atttaccgaa cgaaaaagta ttgcctaagc 3061 acagtttact ttacgagtat ttcacagtgt acaatgaact cacgaaagtt aagtatgtca 3121 ctgagggcat gcgtaaaccc gcctttctaa gcggagaaca gaagaaagca atagtagatc 3181 tgttattcaa gaccaaccgc aaagtgacag ttaagcaatt gaaagaggac tactttaaga 3241 aaattgaatg cttcgattct gtcgagatct ccggggtaga agatcgattt aatgcgtcac 3301 ttggtacgta tcatgacctc ctaaagataa ttaaagataa ggacttcctg gataacgaag 3361 agaatgaaga tatcttagaa gatatagtgt tgactcttac cctctttgaa gatcgggaaa 3421 tgattgagga aagactaaaa acatacgctc acctgttcga cgataaggtt atgaaacagt 3481 taaagaggcg tcgctatacg ggctggggac gattgtcgcg gaaacttatc aacgggataa 3541 gagacaagca aagtggtaaa actattctcg attttctaaa gagcgacggc ttcgccaata 3601 ggaactttat gcagctgatc catgatgact ctttaacctt caaagaggat atacaaaagg 3661 cacaggtttc cggacaaggg gactcattgc acgaacatat tgcgaatctt gctggttcgc 3721 cagccatcaa aaagggcata ctccagacag tcaaagtagt ggatgagcta gttaaggtca 3781 tgggacgtca caaaccggaa aacattgtaa tcgagatggc acgcgaaaat caaacgactc 3841 agaaggggca aaaaaacagt cgagagcgga tgaagagaat agaagagggt attaaagaac 3901 tgggcagcca gatcttaaag gagcatcctg tggaaaatac ccaattgcag aacgagaaac 3961 tttacctcta ttacctacaa aatggaaggg acatgtatgt tgatcaggaa ctggacataa 4021 accgtttatc tgattacgac gtcgatcaca ttgtacccca atcctttttg aaggacgatt 4081 caatcgacaa taaagtgctt acacgctcgg ataagaaccg agggaaaagt gacaatgttc 4141 caagcgagga agtcgtaaag aaaatgaaga actattggcg gcagctccta aatgcgaaac 4201 tgataacgca aagaaagttc gataacttaa ctaaagctga gaggggtggc ttgtctgaac 4261 ttgacaaggc cggatttatt aaacgtcagc tcgtggaaac ccgccaaatc acaaagcatg 4321 ttgcacagat actagattcc cgaatgaata cgaaatacga cgagaacgat aagctgattc 4381 gggaagtcaa agtaatcact ttaaagtcaa aattggtgtc ggacttcaga aaggattttc 4441 aattctataa agttagggag ataaataact accaccatgc gcacgacgct tatcttaatg 4501 ccgtcgtagg gaccgcactc attaagaaat acccgaagct agaaagtgag tttgtgtatg 4561 gtgattacaa agtttatgac gtccgtaaga tgatcgcgaa aagcgaacag gagataggca 4621 aggctacagc caaatacttc ttttattcta acattatgaa tttctttaag acggaaatca 4681 ctctggcaaa cggagagata cgcaaacgac ctttaattga aaccaatggg gagacaggtg 4741 aaatcgtatg ggataagggc cgggacttcg cgacggtgag aaaagttttg tccatgcccc 4801 aagtcaacat agtaaagaaa actgaggtgc agaccggagg gttttcaaag gaatcgattc 4861 ttccaaaaag gaatagtgat aagctcatcg ctcgtaaaaa ggactgggac ccgaaaaagt 4921 acggtggctt cgatagccct acagttgcct attctgtcct agtagtggca aaagttgaga 4981 agggaaaatc caagaaactg aagtcagtca aagaattatt ggggataacg attatggagc 5041 gctcgtcttt tgaaaagaac cccatcgact tccttgaggc gaaaggttac aaggaagtaa 5101 aaaaggatct cataattaaa ctaccaaagt atagtctgtt tgagttagaa aatggccgaa 5161 aacggatgtt ggctagcgcc ggagagcttc aaaaggggaa cgaactcgca ctaccgtcta 5221 aatacgtgaa tttcctgtat ttagcgtccc attacgagaa gttgaaaggt tcacctgaag 5281 ataacgaaca gaagcaactt tttgttgagc agcacaaaca ttatctcgac gaaatcatag 5341 agcaaatttc ggaattcagt aagagagtca tcctagctga tgccaatctg gacaaagtat 5401 taagcgcata caacaagcac agggataaac ccatacgtga gcaggcggaa aatattatcc 5461 atttgtttac tcttaccaac ctcggcgctc cagccgcatt caagtatttt gacacaacga 5521 tagatcgcaa acgatacact tctaccaagg aggtgctaga cgcgacactg attcaccaat 5581 ccatcacggg attatatgaa actcggatag atttgtcaca gcttgggggt gactctggtg 5641 gttctactaa tctgtcagat attattgaaa aggagaccgg taagcaactg gttatccagg 5701 aatccatcct catgctccca gaggaggtgg aagaagtcat tgggaacaag ccggaaagcg 5761 atatactcgt gcacaccgcc tacgacgaga gcaccgacga gaatgtcatg cttctgacta 5821 gcgacgcccc tgaatacaag ccttgggctc tggtcataca ggatagcaac ggtgagaaca 5881 agattaagat gctctctggt ggttctccca agaagaagag gaaagtctaa ccggtcatca 5941 tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca 6001 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 6061 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 6121 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 6181 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgat 6241 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 6301 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagccta 6361 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 6421 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 6481 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 6541 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 6601 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 6661 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 6721 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 6781 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 6841 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 6901 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 6961 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 7021 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 7081 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 7141 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 7201 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 7261 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 7321 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 7381 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 7441 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 7501 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 7561 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 7621 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 7681 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 7741 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 7801 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 7861 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 7921 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 7981 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 8041 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 8101 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 8161 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 8221 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 8281 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 8341 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 8401 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc gatctcccga 8461 tcccctaggg tc //