LOCUS       sequence-226914-        8871 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 8871)
  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 8871)
  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"; journalName: "Nat 
            Biotechnol. 2019 May 20. pii: 10.1038/s41587-019-0134-y. doi: 
            10.1038/s41587-019-0134-y."
COMMENT     Sequence Label: CP1012-ABEmax
FEATURES             Location/Qualifiers
     source          1..8871
                     /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             2785..4500
                     /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             5857..5877
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5886..5903
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5926..5943)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5932..6156
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6227..6243)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6227..6243)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6240..6262)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6251..6267
                     /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(6275..6281,6282..6299,6300..6305))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6320..6341
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6458..6475)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6629..7217)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6709..6728)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7388..8179,8180..8248))
                     /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     8011..8030
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8249..8353)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8432..8451)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8623..8871,1..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 tcagactaca aggtgtacga cgtgcggaag
     1681 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     1741 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     1801 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     1861 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     1921 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     1981 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     2041 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     2101 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     2161 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     2221 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     2281 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     2341 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     2401 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     2461 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     2521 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     2581 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     2641 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     2701 gacctgtctc agctgggagg cgacggcgga tcaggaggct ctggcggttc aggtggatca
     2761 ggcggtagcg gaggttcagg tggtgacaag aagtacagca tcggcctggc catcggcacc
     2821 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     2881 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     2941 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     3001 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     3061 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     3121 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     3181 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     3241 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     3301 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     3361 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     3421 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     3481 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     3541 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     3601 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     3661 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     3721 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     3781 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     3841 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     3901 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     3961 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     4021 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     4081 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     4141 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     4201 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     4261 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     4321 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     4381 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     4441 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     4501 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     4561 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     4621 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     4681 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     4741 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     4801 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     4861 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     4921 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     4981 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     5041 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     5101 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     5161 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
     5221 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     5281 tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac
     5341 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     5401 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     5461 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     5521 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     5581 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     5641 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     5701 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     5761 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggctctggc
     5821 ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtctaa
     5881 ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt
     5941 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     6001 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     6061 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     6121 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     6181 ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     6241 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     6301 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     6361 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6421 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6481 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6541 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6601 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6661 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6721 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6781 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6841 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6901 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6961 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     7021 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     7081 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     7141 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     7201 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     7261 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     7321 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     7381 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     7441 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7501 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7561 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7621 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7681 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7741 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7801 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7861 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7921 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7981 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     8041 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     8101 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     8161 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     8221 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     8281 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     8341 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     8401 gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa
     8461 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     8521 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     8581 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     8641 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     8701 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     8761 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     8821 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat c
//
