LOCUS       sequence-272893-        8277 bp    DNA     circular SYN 14-APR-2025
DEFINITION  A-to-G base editor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8277)
  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 Jul;38(7):883-891. doi: 
            10.1038/s41587-020-0453-z. Epub 2020 Mar 16.
  PUBMED    32433547
REFERENCE   2  (bases 1 to 8277)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41587-020-0453-z"; journalName: "Nat Biotechnol"; date: 
            "2020-07"; volume: "38"; issue: "7"; pages: "883-891"
COMMENT     Sequence Label: CP1041-ABE8e
FEATURES             Location/Qualifiers
     source          1..8277
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             2104..3819
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             5263..5283
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5292..5309
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5332..5349)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5338..5562
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5633..5649)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5633..5649)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5646..5668)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5657..5673
                     /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(join(5681..5687,5688..5705,5706..5711))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5726..5747
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5864..5881)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6035..6623)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6115..6134)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6794..7585,7586..7654))
                     /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     7417..7436
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7655..7759)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7838..7857)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8029..8277,1..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 ggggggtcaa acatcatgaa ctttttcaag
     1081 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     1141 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     1201 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     1261 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     1321 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     1381 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     1441 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     1501 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     1561 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     1621 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     1681 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     1741 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     1801 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     1861 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     1921 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     1981 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     2041 gacggcggat caggaggctc tggcggttca ggtggatcag gcggtagcgg aggttcaggt
     2101 ggtgacaaga agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg
     2161 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     2221 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     2281 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     2341 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     2401 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     2461 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     2521 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     2581 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     2641 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     2701 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     2761 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac
     2821 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     2881 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     2941 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     3001 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     3061 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     3121 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     3181 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     3241 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     3301 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     3361 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     3421 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     3481 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     3541 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     3601 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     3661 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     3721 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     3781 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     3841 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     3901 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     3961 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     4021 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     4081 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     4141 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     4201 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     4261 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     4321 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     4381 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     4441 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     4501 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     4561 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
     4621 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     4681 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     4741 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     4801 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     4861 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     4921 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     4981 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     5041 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     5101 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     5161 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     5221 tctggcggct caaaaagaac cgccgacggc agcgaattcg agcccaagaa gaagaggaaa
     5281 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg
     5341 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     5401 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     5461 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     5521 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     5581 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5641 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5701 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5761 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5821 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5881 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5941 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6001 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6061 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6121 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6181 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6241 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6301 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6361 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6421 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6481 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6541 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6601 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacactc
     6661 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6721 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6781 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6841 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6901 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6961 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7021 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7081 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7141 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7201 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7261 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7321 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7381 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7441 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7501 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7561 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7621 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7681 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7741 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     7801 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat
     7861 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     7921 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     7981 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     8041 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatattgagt
     8101 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     8161 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     8221 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc
//
