LOCUS       sequence-226918-        8874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A-to-G base editor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8874)
  AUTHORS   Huang TP, Zhao KT, Miller SM, Gaudelli NM, Oakes BL, Fellmann C, 
            Savage DF, Liu DR
  TITLE     Circularly permuted and PAM-modified Cas9 variants broaden the 
            targeting scope of base editors.
  JOURNAL   Nat Biotechnol. 2019 May 20. pii: 10.1038/s41587-019-0134-y. doi: 
            10.1038/s41587-019-0134-y.
  PUBMED    31110355
REFERENCE   2  (bases 1 to 8874)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2019 May 20. pii: 10.1038/s41587-019-0134-y. doi: 
            10.1038/s41587-019-0134-y."
COMMENT     Sequence Label: CP1249-ABEmax
FEATURES             Location/Qualifiers
     source          1..8874
                     /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             2074..3792
                     /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             5860..5880
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5889..5906
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5929..5946)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5935..6159
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6230..6246)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6230..6246)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6243..6265)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6254..6270
                     /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(6278..6284,6285..6302,6303..6308))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6323..6344
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6461..6478)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6632..7220)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6712..6731)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7391..8182,8183..8251))
                     /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     8014..8033
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8252..8356)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8435..8454)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8626..8874,1..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 agtcgagttt
      481 agccacgagt attggatgag gcacgcactg accctggcaa agcgagcatg ggatgaaaga
      541 gaagtccccg tgggcgccgt gctggtgcac aacaatagag tgatcggaga gggatggaac
      601 aggccaatcg gccgccacga ccctaccgca cacgcagaga tcatggcact gaggcaggga
      661 ggcctggtca tgcagaatta ccgcctgatc gatgccaccc tgtatgtgac actggagcca
      721 tgcgtgatgt gcgcaggagc aatgatccac agcaggatcg gaagagtggt gttcggagca
      781 cgggacgcca agaccggcgc agcaggctcc ctgatggatg tgctgcacca ccccggcatg
      841 aaccaccggg tggagatcac agagggaatc ctggcagacg agtgcgccgc cctgctgagc
      901 gatttcttta gaatgcggag acaggagatc aaggcccaga agaaggcaca gagctccacc
      961 gactctggag gatctagcgg aggatcctct ggaagcgaga caccaggcac aagcgagtcc
     1021 gccacaccag agagctccgg cggctcctcc ggaggatcct ctgaggtgga gttttcccac
     1081 gagtactgga tgagacatgc cctgaccctg gccaagaggg cacgcgatga gagggaggtg
     1141 cctgtgggag ccgtgctggt gctgaacaat agagtgatcg gcgagggctg gaacagagcc
     1201 atcggcctgc acgacccaac agcccatgcc gaaattatgg ccctgagaca gggcggcctg
     1261 gtcatgcaga actacagact gattgacgcc accctgtacg tgacattcga gccttgcgtg
     1321 atgtgcgccg gcgccatgat ccactctagg atcggccgcg tggtgtttgg cgtgaggaac
     1381 gcaaaaaccg gcgccgcagg ctccctgatg gacgtgctgc actaccccgg catgaatcac
     1441 cgcgtcgaaa ttaccgaggg aatcctggca gatgaatgtg ccgccctgct gtgctatttc
     1501 tttcggatgc ctagacaggt gttcaatgct cagaagaagg cccagagctc caccgactcc
     1561 ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca
     1621 cctgaaagca gcgggggcag cagcgggggg tcacccgagg ataatgagca gaaacagctg
     1681 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     1741 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     1801 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     1861 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     1921 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     1981 acacggatcg acctgtctca gctgggaggc gacggcggat caggaggctc tggcggttca
     2041 ggtggatcag gcggtagcgg aggttcaggt ggtatggaca agaagtacag catcggcctg
     2101 gccatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc
     2161 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga
     2221 gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga
     2281 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag
     2341 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
     2401 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac
     2461 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc
     2521 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg
     2581 atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg
     2641 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag
     2701 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg
     2761 cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc
     2821 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac
     2881 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg
     2941 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
     3001 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac
     3061 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
     3121 attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag
     3181 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg
     3241 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
     3301 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt
     3361 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
     3421 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc
     3481 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag
     3541 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
     3601 aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac
     3661 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg
     3721 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc
     3781 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
     3841 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
     3901 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
     3961 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
     4021 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
     4081 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
     4141 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
     4201 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
     4261 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
     4321 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
     4381 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
     4441 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
     4501 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
     4561 atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac
     4621 gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac
     4681 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc
     4741 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
     4801 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
     4861 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg
     4921 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
     4981 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg
     5041 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
     5101 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc
     5161 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
     5221 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc
     5281 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg
     5341 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
     5401 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag
     5461 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
     5521 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
     5581 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
     5641 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
     5701 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc
     5761 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctcctct
     5821 ggcggctcaa aaagaaccgc cgacggcagc gaattcgagc ccaagaagaa gaggaaagtc
     5881 taaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc
     5941 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     6001 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     6061 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     6121 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     6181 gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     6241 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     6301 agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     6361 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6421 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     6481 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     6541 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     6601 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6661 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6721 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6781 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6841 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6901 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6961 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     7021 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     7081 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     7141 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     7201 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     7261 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     7321 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     7381 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7441 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7501 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7561 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7621 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7681 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     7741 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7801 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7861 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7921 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7981 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     8041 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     8101 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     8161 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     8221 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     8281 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8341 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     8401 atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt
     8461 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     8521 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     8581 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     8641 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     8701 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     8761 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     8821 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatc
//
