LOCUS Exported 8439 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses AID-BE3 in mammalian cells. ACCESSION . VERSION . KEYWORDS AID-BE3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8439) AUTHORS Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, Kim YB, Badran AH, Liu DR TITLE Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. JOURNAL Sci Adv. 2017 Aug 30;3(8):eaao4774. doi: 10.1126/sciadv.aao4774. eCollection 2017 Aug. PUBMED 28875174 REFERENCE 2 (bases 1 to 8439) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8439 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 132..335 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 286..306 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 377..396 /label=T7 /note="T7 promoter, forward primer" promoter 377..395 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 403..412 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 409..1002 /codon_start=1 /gene="AICDA" /product="human activation-induced cytidine deaminase (Dickerson et al., 2003)" /label=AID /translation="MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLD FGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNL SLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWE GLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL" CDS 1051..5151 /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 5164..5412 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5425..5445 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5454..5471 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5494..5511) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5500..5724 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5795..5811) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5795..5811) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5808..5830) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5819..5835 /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(5843..5873) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5888..5909 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6026..6043) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6197..6785) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6277..6296) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6956..7816) /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 7579..7598 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7817..7921) /gene="bla" /label=AmpR promoter primer_bind complement(8000..8019) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 8191..131 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggatagcctc 421 ttgatgaata gacgcaagtt cctgtatcag tttaaaaacg tgagatgggc aaaaggccga 481 cgagagacat atctgtgcta tgtcgttaag cgcagagatt cagccaccag tttctctctc 541 gacttcggct acctgcggaa caagaatggt tgccatgttg agctcctgtt cctgaggtat 601 atcagcgact gggatttgga cccagggcgg tgctataggg tgacatggtt tacctcctgg 661 tcaccttgtt atgactgcgc gcggcatgtt gccgattttc tgagagggaa ccctaacctg 721 tctctgagga tcttcaccgc gcgactgtac ttctgtgagg accggaaagc cgaacccgag 781 ggactgagac gcctccacag agcgggtgtg cagattgcca taatgacctt taaggactac 841 ttctactgct ggaacacctt cgtcgaaaat cacgagcgga ctttcaaggc ttgggaagga 901 ttgcatgaaa acagcgtcag gctttccagg cagcttcgcc gcattcttct cccgttgtac 961 gaggttgatg acctcagaga tgcctttaga acactgggac tgagcggcag cgagactccc 1021 gggacctcag agtccgccac acccgaaagt gataaaaagt attctattgg tttagccatc 1081 ggcactaatt ccgttggatg ggctgtcata accgatgaat acaaagtacc ttcaaagaaa 1141 tttaaggtgt tggggaacac agaccgtcat tcgattaaaa agaatcttat cggtgccctc 1201 ctattcgata gtggcgaaac ggcagaggcg actcgcctga aacgaaccgc tcggagaagg 1261 tatacacgtc gcaagaaccg aatatgttac ttacaagaaa tttttagcaa tgagatggcc 1321 aaagttgacg attctttctt tcaccgtttg gaagagtcct tccttgtcga agaggacaag 1381 aaacatgaac ggcaccccat ctttggaaac atagtagatg aggtggcata tcatgaaaag 1441 tacccaacga tttatcacct cagaaaaaag ctagttgact caactgataa agcggacctg 1501 aggttaatct acttggctct tgcccatatg ataaagttcc gtgggcactt tctcattgag 1561 ggtgatctaa atccggacaa ctcggatgtc gacaaactgt tcatccagtt agtacaaacc 1621 tataatcagt tgtttgaaga gaaccctata aatgcaagtg gcgtggatgc gaaggctatt 1681 cttagcgccc gcctctctaa atcccgacgg ctagaaaacc tgatcgcaca attacccgga 1741 gagaagaaaa atgggttgtt cggtaacctt atagcgctct cactaggcct gacaccaaat 1801 tttaagtcga acttcgactt agctgaagat gccaaattgc agcttagtaa ggacacgtac 1861 gatgacgatc tcgacaatct actggcacaa attggagatc agtatgcgga cttatttttg 1921 gctgccaaaa accttagcga tgcaatcctc ctatctgaca tactgagagt taatactgag 1981 attaccaagg cgccgttatc cgcttcaatg atcaaaaggt acgatgaaca tcaccaagac 2041 ttgacacttc tcaaggccct agtccgtcag caactgcctg agaaatataa ggaaatattc 2101 tttgatcagt cgaaaaacgg gtacgcaggt tatattgacg gcggagcgag tcaagaggaa 2161 ttctacaagt ttatcaaacc catattagag aagatggatg ggacggaaga gttgcttgta 2221 aaactcaatc gcgaagatct actgcgaaag cagcggactt tcgacaacgg tagcattcca 2281 catcaaatcc acttaggcga attgcatgct atacttagaa ggcaggagga tttttatccg 2341 ttcctcaaag acaatcgtga aaagattgag aaaatcctaa cctttcgcat accttactat 2401 gtgggacccc tggcccgagg gaactctcgg ttcgcatgga tgacaagaaa gtccgaagaa 2461 acgattactc catggaattt tgaggaagtt gtcgataaag gtgcgtcagc tcaatcgttc 2521 atcgagagga tgaccaactt tgacaagaat ttaccgaacg aaaaagtatt gcctaagcac 2581 agtttacttt acgagtattt cacagtgtac aatgaactca cgaaagttaa gtatgtcact 2641 gagggcatgc gtaaacccgc ctttctaagc ggagaacaga agaaagcaat agtagatctg 2701 ttattcaaga ccaaccgcaa agtgacagtt aagcaattga aagaggacta ctttaagaaa 2761 attgaatgct tcgattctgt cgagatctcc ggggtagaag atcgatttaa tgcgtcactt 2821 ggtacgtatc atgacctcct aaagataatt aaagataagg acttcctgga taacgaagag 2881 aatgaagata tcttagaaga tatagtgttg actcttaccc tctttgaaga tcgggaaatg 2941 attgaggaaa gactaaaaac atacgctcac ctgttcgacg ataaggttat gaaacagtta 3001 aagaggcgtc gctatacggg ctggggacga ttgtcgcgga aacttatcaa cgggataaga 3061 gacaagcaaa gtggtaaaac tattctcgat tttctaaaga gcgacggctt cgccaatagg 3121 aactttatgc agctgatcca tgatgactct ttaaccttca aagaggatat acaaaaggca 3181 caggtttccg gacaagggga ctcattgcac gaacatattg cgaatcttgc tggttcgcca 3241 gccatcaaaa agggcatact ccagacagtc aaagtagtgg atgagctagt taaggtcatg 3301 ggacgtcaca aaccggaaaa cattgtaatc gagatggcac gcgaaaatca aacgactcag 3361 aaggggcaaa aaaacagtcg agagcggatg aagagaatag aagagggtat taaagaactg 3421 ggcagccaga tcttaaagga gcatcctgtg gaaaataccc aattgcagaa cgagaaactt 3481 tacctctatt acctacaaaa tggaagggac atgtatgttg atcaggaact ggacataaac 3541 cgtttatctg attacgacgt cgatcacatt gtaccccaat cctttttgaa ggacgattca 3601 atcgacaata aagtgcttac acgctcggat aagaaccgag ggaaaagtga caatgttcca 3661 agcgaggaag tcgtaaagaa aatgaagaac tattggcggc agctcctaaa tgcgaaactg 3721 ataacgcaaa gaaagttcga taacttaact aaagctgaga ggggtggctt gtctgaactt 3781 gacaaggccg gatttattaa acgtcagctc gtggaaaccc gccaaatcac aaagcatgtt 3841 gcacagatac tagattcccg aatgaatacg aaatacgacg agaacgataa gctgattcgg 3901 gaagtcaaag taatcacttt aaagtcaaaa ttggtgtcgg acttcagaaa ggattttcaa 3961 ttctataaag ttagggagat aaataactac caccatgcgc acgacgctta tcttaatgcc 4021 gtcgtaggga ccgcactcat taagaaatac ccgaagctag aaagtgagtt tgtgtatggt 4081 gattacaaag tttatgacgt ccgtaagatg atcgcgaaaa gcgaacagga gataggcaag 4141 gctacagcca aatacttctt ttattctaac attatgaatt tctttaagac ggaaatcact 4201 ctggcaaacg gagagatacg caaacgacct ttaattgaaa ccaatgggga gacaggtgaa 4261 atcgtatggg ataagggccg ggacttcgcg acggtgagaa aagttttgtc catgccccaa 4321 gtcaacatag taaagaaaac tgaggtgcag accggagggt tttcaaagga atcgattctt 4381 ccaaaaagga atagtgataa gctcatcgct cgtaaaaagg actgggaccc gaaaaagtac 4441 ggtggcttcg atagccctac agttgcctat tctgtcctag tagtggcaaa agttgagaag 4501 ggaaaatcca agaaactgaa gtcagtcaaa gaattattgg ggataacgat tatggagcgc 4561 tcgtcttttg aaaagaaccc catcgacttc cttgaggcga aaggttacaa ggaagtaaaa 4621 aaggatctca taattaaact accaaagtat agtctgtttg agttagaaaa tggccgaaaa 4681 cggatgttgg ctagcgccgg agagcttcaa aaggggaacg aactcgcact accgtctaaa 4741 tacgtgaatt tcctgtattt agcgtcccat tacgagaagt tgaaaggttc acctgaagat 4801 aacgaacaga agcaactttt tgttgagcag cacaaacatt atctcgacga aatcatagag 4861 caaatttcgg aattcagtaa gagagtcatc ctagctgatg ccaatctgga caaagtatta 4921 agcgcataca acaagcacag ggataaaccc atacgtgagc aggcggaaaa tattatccat 4981 ttgtttactc ttaccaacct cggcgctcca gccgcattca agtattttga cacaacgata 5041 gatcgcaaac gatacacttc taccaaggag gtgctagacg cgacactgat tcaccaatcc 5101 atcacgggat tatatgaaac tcggatagat ttgtcacagc ttgggggtga ctctggtggt 5161 tctactaatc tgtcagatat tattgaaaag gagaccggta agcaactggt tatccaggaa 5221 tccatcctca tgctcccaga ggaggtggaa gaagtcattg ggaacaagcc ggaaagcgat 5281 atactcgtgc acaccgccta cgacgagagc accgacgaga atgtcatgct tctgactagc 5341 gacgcccctg aatacaagcc ttgggctctg gtcatacagg atagcaacgg tgagaacaag 5401 attaagatgc tctctggtgg ttctcccaag aagaagagga aagtctaacc ggtcatcatc 5461 accatcacca ttgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 5521 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 5581 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 5641 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 5701 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgatac 5761 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 5821 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctagg 5881 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 5941 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 6001 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 6061 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 6121 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 6181 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 6241 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 6301 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 6361 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 6421 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 6481 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 6541 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 6601 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc 6661 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 6721 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 6781 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 6841 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 6901 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 6961 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 7021 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 7081 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 7141 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 7201 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 7261 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 7321 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 7381 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 7441 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 7501 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 7561 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 7621 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 7681 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 7741 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 7801 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 7861 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 7921 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatcga tctcccgatc 7981 ccctagggtc gactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc 8041 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa 8101 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc 8161 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt 8221 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 8281 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 8341 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 8401 tacggtaaac tgcccacttg gcagtacatc aagtgtatc //