LOCUS Exported 8601 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses SaBE4-Gam in mammalian cells. ACCESSION . VERSION . KEYWORDS SaBE4-Gam SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8601) 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 8601) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8601 /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 979..1662 /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="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL WATGLK" CDS 4924..4944 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4951..5016 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 5047..5295 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5326..5574 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5587..5607 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5616..5633 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5656..5673) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5662..5886 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5957..5973) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5957..5973) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5970..5992) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5981..5997 /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(6005..6035) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6050..6071 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6188..6205) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6359..6947) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6439..6458) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7118..7978) /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 7741..7760 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7979..8083) /gene="bla" /label=AmpR promoter primer_bind complement(8162..8181) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 8353..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 ggctaaacca 421 gcaaaacgta tcaagagtgc cgcagcggct tatgtgccac aaaaccgcga tgcggtgatt 481 accgatatta aacgcatcgg ggatttacag cgcgaagcat cacgtctgga aacggaaatg 541 aatgatgcca tcgcggaaat tacggagaaa tttgcggccc ggattgcacc gattaaaacc 601 gatattgaaa ccctttcaaa aggcgttcag ggatggtgtg aagcgaaccg cgacgaactg 661 acgaacggcg gcaaagtgaa gacggcgaat cttgtcaccg gtgatgtatc gtggcgggtc 721 cgtccaccat cagtaagtat tcgtggtatg gatgcagtga tggaaacgct ggagcgtctt 781 ggcctgcaac gctttattcg cacgaagcag gaaatcaaca aggaagcgat tttactggaa 841 ccgaaagcgg tcgcaggcgt tgccggaatt acagttaaat caggcattga ggatttttct 901 attattccat ttgaacagga agccggtatt agcggcagcg agactcccgg gacctcagag 961 tccgctacac ccgaaagtag ctcagagact ggcccagtgg ctgtggaccc cacattgaga 1021 cggcggatcg agccccatga gtttgaggta ttcttcgatc cgagagagct ccgcaaggag 1081 acctgcctgc tttacgaaat taattggggg ggccggcact ccatttggcg acatacatca 1141 cagaacacta acaagcacgt cgaagtcaac ttcatcgaga agttcacgac agaaagatat 1201 ttctgtccga acacaaggtg cagcattacc tggtttctca gctggagccc atgcggcgaa 1261 tgtagtaggg ccatcactga attcctgtca aggtatcccc acgtcactct gtttatttac 1321 atcgcaaggc tgtaccacca cgctgacccc cgcaatcgac aaggcctgcg ggatttgatc 1381 tcttcaggtg tgactatcca aattatgact gagcaggagt caggatactg ctggagaaac 1441 tttgtgaatt atagcccgag taatgaagcc cactggccta ggtatcccca tctgtgggta 1501 cgactgtacg ttcttgaact gtactgcatc atactgggcc tgcctccttg tctcaacatt 1561 ctgagaagga agcagccaca gctgacattc tttaccatcg ctcttcagtc ttgtcattac 1621 cagcgactgc ccccacacat tctctgggcc accgggttga aatctggtgg ttcttctggt 1681 ggttctagcg gcagcgagac tcccgggacc tcagagtccg ccacacccga aagttctggt 1741 ggttcttctg gtggttctgg gaaacggaac tacatcctgg ggcttgccat tgggataacc 1801 agcgttggct acggaattat tgattatgag acacgcgatg tgattgacgc cggggttagg 1861 ctgttcaaag aggccaacgt tgaaaacaac gagggaagac ggagtaagcg cggagcaaga 1921 agactcaagc gcagacggag acatcggatt cagagggtga aaaagctgct cttcgattac 1981 aatctcctga ccgatcatag tgagctgagc ggaatcaacc cctacgaggc gcgagtgaaa 2041 gggctttccc agaagctgtc cgaagaggag ttctccgccg cgttgctgca cctggccaaa 2101 cggagggggg ttcacaatgt aaacgaagtg gaggaggaca cgggcaatga acttagtacg 2161 aaagaacaga tcagtaggaa ctctaaggct ctcgaagaga aatacgtcgc tgagttgcag 2221 cttgagagac tgaaaaaaga cggcgaagta cgcggatcta ttaataggtt caagacttca 2281 gattacgtaa aggaagccaa gcagctcctg aaagtacaga aagcgtacca tcagctcgat 2341 cagagcttca tcgataccta catagatttg ctggagacac ggaggacata ctacgagggc 2401 ccaggggaag gatctccttt tgggtggaag gacatcaagg aatggtacga gatgcttatg 2461 ggacattgta catattttcc ggaggagctc aggagcgtca agtacgccta caatgccgac 2521 ctgtacaatg ccctcaatga cctcaataac ctcgtgatta ccagggacga gaacgagaag 2581 ctggagtact atgaaaagtt ccagattatc gagaatgtgt ttaagcagaa gaagaagccg 2641 acacttaagc agattgcaaa ggaaatcctc gtgaatgagg aagatatcaa gggatacaga 2701 gtgacaagta caggcaagcc cgagttcaca aatctgaagg tgtaccacga tattaaggac 2761 ataaccgcac gaaaggagat aatcgaaaac gctgagctcc tcgatcagat cgcaaaaatt 2821 cttaccatct accagtctag tgaggacatt caggaggaac tgactaatct gaacagtgag 2881 ctcacccaag aggaaattga gcagatttca aacctgaaag gctacaccgg gacgcacaat 2941 ctgagcctca aagcaatcaa cctcattctg gatgaacttt ggcacacaaa tgacaaccaa 3001 attgccatat tcaaccgcct gaaactggtg ccaaaaaaag tggatctgtc acagcaaaag 3061 gaaatcccta caaccttggt tgacgatttt attctgtccc ccgttgtcaa gcggagcttc 3121 atccagtcaa tcaaggtgat caatgccatc attaaaaaat acggattgcc aaacgatata 3181 attatcgagc ttgcacgaga gaagaactca aaggacgccc agaagatgat taacgaaatg 3241 cagaagcgca accgccagac aaacgaacgc atagaggaaa ttataagaac aaccggcaaa 3301 gagaatgcca agtatctgat cgagaaaatc aagctgcacg acatgcaaga aggcaagtgc 3361 ctgtactctc tggaagctat cccactcgaa gatctgctga ataatccatt caattacgag 3421 gtggaccaca tcatccctag atccgtaagc tttgacaatt ccttcaataa caaagttctg 3481 gttaaacagg aggaaaattc taaaaaaggg aaccggaccc cgttccagta cctgagctcc 3541 agtgacagca agattagcta cgagactttt aagaaacata ttctgaatct ggccaaaggc 3601 aaaggcagga tcagcaagac caagaaggag tacctcctcg aagaacgcga cattaacaga 3661 tttagtgtgc agaaagattt catcaaccga aaccttgtcg atactcggta cgccacgaga 3721 ggcctgatga atctcctcag gagctacttc cgcgtcaata atctggacgt taaagtcaag 3781 agcataaatg ggggattcac cagctttctg aggagaaagt ggaagtttaa gaaggaacga 3841 aacaaaggat acaagcacca tgctgaggat gctttgatca tcgctaacgc ggactttatc 3901 tttaaggaat ggaaaaagct ggataaggca aagaaagtga tggaaaacca gatgttcgag 3961 gagaagcagg cagagtcaat gcctgagatc gagacagagc aggaatacaa ggaaattttc 4021 atcacccctc atcagattaa acacataaag gacttcaaag actataaata ctctcatagg 4081 gtggacaaaa aacccaatcg cgagctcatt aatgacaccc tgtactcaac acggaaggat 4141 gataaaggta ataccttgat tgtgaataat cttaatggat tgtatgacaa agataacgac 4201 aagctcaaga agctgatcaa caagtctcca gagaagctcc ttatgtatca ccacgaccca 4261 cagacttatc agaaattgaa actgatcatg gagcaatacg gggatgagaa gaacccactc 4321 tacaaatatt atgaggaaac aggtaattac ctgaccaagt actccaagaa ggataacgga 4381 ccagtgatca aaaagataaa gtactatggc aacaaactta atgcgcattt ggacataact 4441 gacgattacc ccaattctcg aaacaaggtt gtgaagctct ccctgaagcc ttatagattt 4501 gacgtgtacc tggataatgg ggtttataaa ttcgtcaccg tgaaaaatct ggacgtgatc 4561 aaaaaggaga actattatga agtaaactca aagtgctatg aggaggcgaa gaagctgaag 4621 aagatctcca atcaggccga gttcatcgct tccttctata ataacgatct catcaagatc 4681 aatggagagc tttatcgcgt cattggtgtg aacaatgact tgctgaacag gatcgaagtc 4741 aatatgatag acattaccta ccgggagtat ctcgaaaaca tgaatgataa acggccgcct 4801 cgcatcatca agacaatcgc atctaaaact cagtcaataa aaaagtactc taccgatatc 4861 ctggggaatc tctatgaagt gaagtcaaag aagcacccac aaatcattaa aaaaggtgga 4921 tcccccaaga agaagaggaa agtctcgagc gactacaaag accatgacgg tgattataaa 4981 gatcatgaca tcgattacaa ggatgacgat gacaagtctg gtggttctgg aggatctggt 5041 ggttctacta atctgtcaga tattattgaa aaggagaccg gtaagcaact ggttatccag 5101 gaatccatcc tcatgctccc agaggaggtg gaagaagtca ttgggaacaa gccggaaagc 5161 gatatactcg tgcacaccgc ctacgacgag agcaccgacg agaatgtcat gcttctgact 5221 agcgacgccc ctgaatacaa gccttgggct ctggtcatac aggatagcaa cggtgagaac 5281 aagattaaga tgctctctgg tggttctgga ggatctggtg gttctactaa tctgtcagat 5341 attattgaaa aggagaccgg taagcaactg gttatccagg aatccatcct catgctccca 5401 gaggaggtgg aagaagtcat tgggaacaag ccggaaagcg atatactcgt gcacaccgcc 5461 tacgacgaga gcaccgacga gaatgtcatg cttctgacta gcgacgcccc tgaatacaag 5521 ccttgggctc tggtcataca ggatagcaac ggtgagaaca agattaagat gctctctggt 5581 ggttctccca agaagaagag gaaagtctaa ccggtcatca tcaccatcac cattgagttt 5641 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 5701 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 5761 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 5821 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 5881 ctatggcttc tgaggcggaa agaaccagct ggggctcgat accgtcgacc tctagctaga 5941 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 6001 cacacaacat acgagccgga agcataaagt gtaaagccta gggtgcctaa tgagtgagct 6061 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 6121 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 6181 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 6241 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 6301 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 6361 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 6421 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 6481 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 6541 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 6601 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 6661 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 6721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 6781 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 6841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 6901 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 6961 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 7021 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 7081 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 7141 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 7201 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 7261 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 7321 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 7381 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 7441 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 7501 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 7561 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 7621 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 7681 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 7741 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 7801 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 7861 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 7921 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 7981 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 8041 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 8101 tgccacctga cgtcgacgga tcgggagatc gatctcccga tcccctaggg tcgactctca 8161 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg 8221 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca 8281 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca 8341 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 8401 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 8461 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 8521 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 8581 tggcagtaca tcaagtgtat c //