LOCUS       Exported                8280 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  A-to-G base editor.
ACCESSION   .
VERSION     .
KEYWORDS    CP1028-ABE8e
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8280)
  AUTHORS   Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, 
            Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR
  TITLE     Phage-assisted evolution of an adenine base editor with improved Cas
            domain compatibility and activity
  JOURNAL   Nat Biotechnol (2020)
REFERENCE   2  (bases 1 to 8280)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8280
                     /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             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2143..3861
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             5266..5286
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5295..5312
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5335..5352)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5341..5565
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5636..5652)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5636..5652)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5649..5671)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5660..5676
                     /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(5684..5714)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5729..5750
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5867..5884)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6038..6626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6118..6137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6797..7657)
                     /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     7420..7439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7658..7762)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7841..7860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        8032..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 gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt
      481 tcccacgagt actggatgag acatgccctg accctggcca agagggcacg ggatgagagg
      541 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac
      601 agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc
      661 ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct
      721 tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg
      781 aggaactcaa aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg
      841 aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc
      901 gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc
      961 aactccggag gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc
     1021 gcaacacctg aaagcagcgg gggcagcagc ggggggtcag aaatcggcaa ggctaccgcc
     1081 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     1141 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg
     1201 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc
     1261 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     1321 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     1381 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     1441 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     1501 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     1561 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     1621 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     1681 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     1741 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc
     1801 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac
     1861 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc
     1921 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     1981 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     2041 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acggcggatc aggaggctct
     2101 ggcggttcag gtggatcagg cggtagcgga ggttcaggtg gtatggacaa gaagtacagc
     2161 atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag
     2221 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac
     2281 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga
     2341 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc
     2401 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg
     2461 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg
     2521 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc
     2581 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc
     2641 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc
     2701 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg
     2761 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc
     2821 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg
     2881 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg
     2941 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac
     3001 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg
     3061 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac
     3121 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag
     3181 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga
     3241 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc
     3301 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac
     3361 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag
     3421 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc
     3481 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc
     3541 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct
     3601 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag
     3661 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa
     3721 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag
     3781 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag
     3841 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg
     3901 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc
     3961 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt
     4021 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa
     4081 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg
     4141 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac
     4201 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag
     4261 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat
     4321 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag
     4381 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag
     4441 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag
     4501 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg
     4561 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag
     4621 gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt
     4681 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag
     4741 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg
     4801 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc
     4861 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag
     4921 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat
     4981 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc
     5041 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac
     5101 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc
     5161 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag
     5221 cagtctggcg gctcaaaaag aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg
     5281 aaagtctaac cggtcatcat caccatcacc attgagttta aacccgctga tcagcctcga
     5341 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     5401 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     5461 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     5521 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     5581 gaaccagctg gggctcgata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     5641 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5701 gcataaagtg taaagcctag ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5761 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5821 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5881 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5941 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6001 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6061 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6121 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6181 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6241 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6301 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6361 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6421 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6481 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6541 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6601 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgaca
     6661 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6721 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6781 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6841 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6901 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6961 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7021 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7081 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7141 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7201 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7261 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7321 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7381 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7441 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7501 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7561 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7621 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7681 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7741 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     7801 cgggagatcg atctcccgat cccctagggt cgactctcag tacaatctgc tctgatgccg
     7861 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     7921 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
     7981 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
     8041 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatattg
     8101 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     8161 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     8221 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
//
