LOCUS Exported 8532 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses VQR-BE3 in mammalian cells. ACCESSION . VERSION . KEYWORDS pBK-VQR-BE3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8532) AUTHORS Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR TITLE Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. JOURNAL Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3803. PUBMED 28191901 REFERENCE 2 (bases 1 to 8532) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8532 /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" CDS 409..1095 /codon_start=1 /product="cytidine deaminase (C to U editing enzyme) from rat" /label=APOBEC-1 /note="can use ssDNA as a substrate (Komor et al., 2016)" /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI LWATGLK" CDS 1144..5244 /codon_start=1 /product="Streptococcus pyogenes Cas9 endonuclease with the D1135V/R1335Q/T1337R mutations" /label=Cas9 VQR /note="has an altered recognition specificity for the protospacer adjacent motif (PAM) (Kleinstiver et al., 2015)" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5257..5505 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5518..5538 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5547..5564 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5587..5604) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5593..5817 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5888..5904) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5888..5904) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5901..5923) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5912..5928 /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(5936..5966) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5981..6002 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6119..6136) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6290..6878) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6370..6389) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7049..7909) /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 7672..7691 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7910..8014) /gene="bla" /label=AmpR promoter primer_bind complement(8093..8112) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 8284..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 gagctcagag 421 actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 481 gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 541 gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 601 aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 661 acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 721 tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 781 ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 841 actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 901 gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 961 atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1021 ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1081 gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1141 agtgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 1201 ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 1261 cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 1321 gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 1381 tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 1441 ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 1501 aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 1561 aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 1621 atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 1681 gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 1741 ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 1801 cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 1861 cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 1921 gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 1981 caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 2041 ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 2101 atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 2161 cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 2221 ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 2281 gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 2341 aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 2401 gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 2461 gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 2521 cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 2581 gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 2641 aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 2701 tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 2761 agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 2821 gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 2881 tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 2941 attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 3001 ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 3061 cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 3121 cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 3181 gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 3241 tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 3301 cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 3361 gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 3421 atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 3481 atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 3541 gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 3601 gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 3661 attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 3721 gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 3781 aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 3841 actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 3901 ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 3961 acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 4021 aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 4081 taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 4141 tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 4201 atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 4261 aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 4321 cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 4381 gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 4441 cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 4501 gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgtgagccc tacagttgcc 4561 tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 4621 aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 4681 ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 4741 tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 4801 caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 4861 cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 4921 cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 4981 atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 5041 cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 5101 ccagccgcat tcaagtattt tgacacaacg atagatcgca aacagtacag atctaccaag 5161 gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 5221 gatttgtcac agcttggggg tgactctggt ggttctacta atctgtcaga tattattgaa 5281 aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5341 gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5401 agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5461 ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5521 aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 5581 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5641 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5701 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5761 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 5821 ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 5881 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 5941 tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 6001 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6061 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6121 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6181 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6241 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6301 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6361 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6421 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6481 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 6541 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6601 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6661 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6721 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6781 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6841 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6901 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6961 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7021 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7081 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7141 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7201 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7261 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7321 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7381 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7441 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7501 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7561 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7621 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7681 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7741 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7801 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7861 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7921 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7981 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8041 acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 8101 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 8161 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 8221 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 8281 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8341 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8401 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8461 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8521 atcaagtgta tc //