LOCUS       sequence-320176-        8313 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian CG-to-GC base editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8313)
  AUTHORS   Koblan LW, Arbab M, Shen MW, Hussmann JA, Anzalone AV, Doman JL, 
            Newby GA, Yang D, Mok B, Replogle JM, Xu A, Sisley TA, Weissman JS, 
            Adamson B, Liu DR
  TITLE     Efficient C*G-to-G*C base editors developed using CRISPRi screens, 
            target-library analysis, and machine learning.
  JOURNAL   Nat Biotechnol. 2021 Jun 28. pii: 10.1038/s41587-021-00938-z. doi: 
            10.1038/s41587-021-00938-z.
  PUBMED    34183861
REFERENCE   2  (bases 1 to 8313)
  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. 2021 Jun 28. pii: 10.1038/s41587-021-00938-z. doi: 
            10.1038/s41587-021-00938-z."
COMMENT     Sequence Label: pCMV_eA3A-NG-nCas9
FEATURES             Location/Qualifiers
     source          1..8313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        110..489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        490..693
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     644..664
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     735..754
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        735..753
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             803..823
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1514..5614
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5657..5677
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5686..5703
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5726..5743)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5732..5956
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6027..6043)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6027..6043)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6040..6062)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6051..6067
                     /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(6075..6081,6082..6099,6100..6105))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6120..6141
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6258..6275)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6429..7017)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6509..6528)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7188..7979,7980..8048))
                     /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     7811..7830
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8049..8153)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8232..8251)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
       61 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      481 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      541 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      601 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      661 cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag
      721 agatccgcgg ccgctaatac gactcactat agggagagcc gccaccatga aacggacagc
      781 cgacggaagc gagttcgagt caccaaagaa gaagcggaaa gtcgaggcaa gcccagcaag
      841 cggcccacgg cacctgatgg acccccacat tttcaccagc aacttcaaca acggaatcgg
      901 acgacacaag acctacctgt gctatgaggt ggagaggctg gacaacggca catccgtgaa
      961 gatggatcag cacagaggct tcctgcacgg ccaggccaag aatctgctgt gcggcttcta
     1021 cggccggcac gcagagctgc gctttctgga cctggtgcca tctctgcagc tggatcccgc
     1081 ccagatctat agggtgacct ggttcatcag ctggtcccct tgctttagct ggggatgtgc
     1141 aggagaggtg agggccttcc tgcaggagaa cacacacgtg aggctgagaa tctttgccgc
     1201 ccggatctac gactatgatc ccctgtacaa ggaggccctg cagatgctga gggacgcagg
     1261 agcccaggtg tctatcatga cctatgatga gttcaagcac tgctgggaca catttgtgga
     1321 tcaccagggc tgtccctttc agccttggga cggcctggac gagcattccc aggcactgag
     1381 cggacggctg agggcaattc tgcagaacca gggcaattct ggcggatcta gcggaggatc
     1441 ctctggcagc gagacaccag gaacaagcga gtcagcaaca ccagagagca gtggcggcag
     1501 cagcggcggc agcgacaaga agtacagcat cggcctggcc atcggcacca actctgtggg
     1561 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
     1621 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
     1681 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     1741 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     1801 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     1861 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     1921 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     1981 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     2041 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     2101 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     2161 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     2221 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     2281 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     2341 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     2401 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     2461 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     2521 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     2581 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     2641 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     2701 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     2761 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     2821 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     2881 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     2941 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     3001 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
     3061 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
     3121 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
     3181 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
     3241 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     3301 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     3361 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     3421 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     3481 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     3541 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     3601 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     3661 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     3721 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     3781 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     3841 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     3901 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     3961 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     4021 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     4081 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     4141 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     4201 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     4261 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     4321 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     4381 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     4441 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     4501 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     4561 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     4621 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     4681 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     4741 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
     4801 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc aggcccaaga ggaacagcga
     4861 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgtcagccc
     4921 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     4981 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     5041 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     5101 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     5161 cagattcctg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     5221 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     5281 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     5341 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     5401 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     5461 tctgggagcc cctagggcct tcaagtactt tgacaccacc atcgaccgga aggtgtacag
     5521 gagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
     5581 gacacggatc gacctgtctc agctgggagg tgactctggc ggctcaaaaa gaaccgccga
     5641 cggcagcgaa ttcgagccca agaagaagag gaaagtctaa ccggtcatca tcaccatcac
     5701 cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5761 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5821 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5881 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     5941 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgat accgtcgacc
     6001 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     6061 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagccta gggtgcctaa
     6121 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     6181 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg gaagaggcgg tttgcgtatt
     6241 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     6301 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     6361 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     6421 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     6481 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6541 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6601 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6661 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6721 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6781 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6841 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6901 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6961 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     7021 gatcctttga tcttttctac ggggtctgac actcagtgga acgaaaactc acgttaaggg
     7081 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     7141 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     7201 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     7261 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     7321 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     7381 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     7441 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     7501 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     7561 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7621 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7681 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7741 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7801 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7861 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7921 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7981 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     8041 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     8101 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     8161 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc gatctcccga tcccctaggg
     8221 tcgactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     8281 tgtgtgttgg aggtcgctga gtagtgcgcg agc
//
