LOCUS       sequence-241016-        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"
     enhancer        95..474
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        475..678
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     629..649
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     720..739
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        720..738
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             788..808
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2417..4135
                     /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             6203..6223
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6232..6249
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6272..6289)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6278..6502
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6573..6589)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6573..6589)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6586..6608)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6597..6613
                     /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(6621..6627,6628..6645,6646..6651))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6666..6687
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6804..6821)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6975..7563)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7055..7074)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7734..8525,8526..8594))
                     /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     8357..8376
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8595..8699)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8778..8797)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
       61 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
      121 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
      181 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      241 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      301 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
      361 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      421 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
      481 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
      541 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
      601 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
      661 gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc cgcggccgct
      721 aatacgactc actataggga gagccgccac catgaaacgg acagccgacg gaagcgagtt
      781 cgagtcacca aagaagaagc ggaaagtctc tgaagtcgag tttagccacg agtattggat
      841 gaggcacgca ctgaccctgg caaagcgagc atgggatgaa agagaagtcc ccgtgggcgc
      901 cgtgctggtg cacaacaata gagtgatcgg agagggatgg aacaggccaa tcggccgcca
      961 cgaccctacc gcacacgcag agatcatggc actgaggcag ggaggcctgg tcatgcagaa
     1021 ttaccgcctg atcgatgcca ccctgtatgt gacactggag ccatgcgtga tgtgcgcagg
     1081 agcaatgatc cacagcagga tcggaagagt ggtgttcgga gcacgggacg ccaagaccgg
     1141 cgcagcaggc tccctgatgg atgtgctgca ccaccccggc atgaaccacc gggtggagat
     1201 cacagaggga atcctggcag acgagtgcgc cgccctgctg agcgatttct ttagaatgcg
     1261 gagacaggag atcaaggccc agaagaaggc acagagctcc accgactctg gaggatctag
     1321 cggaggatcc tctggaagcg agacaccagg cacaagcgag tccgccacac cagagagctc
     1381 cggcggctcc tccggaggat cctctgaggt ggagttttcc cacgagtact ggatgagaca
     1441 tgccctgacc ctggccaaga gggcacgcga tgagagggag gtgcctgtgg gagccgtgct
     1501 ggtgctgaac aatagagtga tcggcgaggg ctggaacaga gccatcggcc tgcacgaccc
     1561 aacagcccat gccgaaatta tggccctgag acagggcggc ctggtcatgc agaactacag
     1621 actgattgac gccaccctgt acgtgacatt cgagccttgc gtgatgtgcg ccggcgccat
     1681 gatccactct aggatcggcc gcgtggtgtt tggcgtgagg aacgcaaaaa ccggcgccgc
     1741 aggctccctg atggacgtgc tgcactaccc cggcatgaat caccgcgtcg aaattaccga
     1801 gggaatcctg gcagatgaat gtgccgccct gctgtgctat ttctttcgga tgcctagaca
     1861 ggtgttcaat gctcagaaga aggcccagag ctccaccgac tccggaggat ctagcggagg
     1921 ctcctctggc tctgagacac ctggcacaag cgagagcgca acacctgaaa gcagcggggg
     1981 cagcagcggg gggtcacccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     2041 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     2101 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     2161 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     2221 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     2281 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     2341 tcagctggga ggcgacggcg gatcaggagg ctctggcggt tcaggtggat caggcggtag
     2401 cggaggttca ggtggtatgg acaagaagta cagcatcggc ctggccatcg gcaccaactc
     2461 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct
     2521 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag
     2581 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg
     2641 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga
     2701 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg
     2761 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat
     2821 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta
     2881 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa
     2941 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct
     3001 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag
     3061 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa
     3121 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa
     3181 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct
     3241 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa
     3301 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc
     3361 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct
     3421 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag
     3481 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt
     3541 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag
     3601 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca
     3661 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga
     3721 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct
     3781 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc
     3841 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat
     3901 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta
     3961 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag
     4021 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac
     4081 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt
     4141 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca
     4201 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat
     4261 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg
     4321 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag
     4381 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc
     4441 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
     4501 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
     4561 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
     4621 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
     4681 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
     4741 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
     4801 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     4861 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     4921 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
     4981 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
     5041 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     5101 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     5161 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     5221 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     5281 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     5341 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
     5401 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt
     5461 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
     5521 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     5581 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     5641 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     5701 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     5761 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
     5821 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     5881 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     5941 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     6001 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     6061 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     6121 cctgtacctg gccagccact atgagaagct gaagggctcc tctggcggct caaaaagaac
     6181 cgccgacggc agcgaattcg agcccaagaa gaagaggaaa gtctaaccgg tcatcatcac
     6241 catcaccatt gagtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     6301 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     6361 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     6421 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     6481 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctcgataccg
     6541 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     6601 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctagggt
     6661 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6721 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6781 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     6841 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     6901 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     6961 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     7021 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     7081 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     7141 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     7201 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     7261 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     7321 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     7381 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     7441 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     7501 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     7561 caagaagatc ctttgatctt ttctacgggg tctgacactc agtggaacga aaactcacgt
     7621 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     7681 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     7741 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     7801 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     7861 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     7921 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     7981 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     8041 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     8101 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     8161 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     8221 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     8281 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     8341 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     8401 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     8461 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     8521 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     8581 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     8641 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     8701 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc tcccgatccc
     8761 ctagggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     8821 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc taca
//
