LOCUS       sequence-320366-        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 T31A-NG-nCas9
FEATURES             Location/Qualifiers
     source          1..8313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             694..714
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1405..5505
                     /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             5548..5568
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5577..5594
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5617..5634)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5623..5847
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5918..5934)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5918..5934)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5931..5953)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5942..5958
                     /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(5966..5972,5973..5990,5991..5996))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6011..6032
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6149..6166)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6320..6908)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6400..6419)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7079..7870,7871..7939))
                     /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     7702..7721
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7940..8044)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8123..8142)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc cgccaccatg
      661 aaacggacag ccgacggaag cgagttcgag tcaccaaaga agaagcggaa agtcgaggca
      721 agcccagcaa gcggcccacg gcacctgatg gacccccaca ttttcaccag caacttcaac
      781 aacggaatcg gacgacacaa ggcctacctg tgctatgagg tggagaggct ggacaacggc
      841 acatccgtga agatggatca gcacagaggc ttcctgcacg gccaggccaa gaatctgctg
      901 tgcggcttct acggccggca cgcagagctg cgctttctgg acctggtgcc atctctgcag
      961 ctggatcccg cccagatcta tagggtgacc tggttcatca gctggtcccc ttgctttagc
     1021 tggggatgtg caggagaggt gagggccttc ctgcaggaga acacacacgt gaggctgaga
     1081 atctttgccg cccggatcta cgactatgat cccctgtaca aggaggccct gcagatgctg
     1141 agggacgcag gagcccaggt gtctatcatg acctatgatg agttcaagca ctgctgggac
     1201 acatttgtgg atcaccaggg ctgtcccttt cagccttggg acggcctgga cgagcattcc
     1261 caggcactga gcggacggct gagggcaatt ctgcagaacc agggcaattc tggcggatct
     1321 agcggaggat cctctggcag cgagacacca ggaacaagcg agtcagcaac accagagagc
     1381 agtggcggca gcagcggcgg cagcgacaag aagtacagca tcggcctggc catcggcacc
     1441 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     1501 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     1561 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     1621 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     1681 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     1741 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     1801 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     1861 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     1921 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     1981 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     2041 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     2101 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     2161 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     2221 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     2281 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     2341 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     2401 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     2461 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     2521 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     2581 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     2641 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     2701 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     2761 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     2821 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     2881 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     2941 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     3001 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     3061 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     3121 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     3181 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     3241 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     3301 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     3361 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     3421 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     3481 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     3541 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     3601 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     3661 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     3721 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     3781 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
     3841 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     3901 tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac
     3961 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     4021 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     4081 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     4141 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     4201 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     4261 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     4321 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     4381 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
     4441 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
     4501 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
     4561 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
     4621 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
     4681 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat caggcccaag
     4741 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
     4801 ttcgtcagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
     4861 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
     4921 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
     4981 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
     5041 ctggcctctg ccagattcct gcagaaggga aacgaactgg ccctgccctc caaatatgtg
     5101 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
     5161 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
     5221 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
     5281 tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt
     5341 accctgacca atctgggagc ccctagggcc ttcaagtact ttgacaccac catcgaccgg
     5401 aaggtgtaca ggagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
     5461 ggcctgtacg agacacggat cgacctgtct cagctgggag gtgactctgg cggctcaaaa
     5521 agaaccgccg acggcagcga attcgagccc aagaagaaga ggaaagtcta accggtcatc
     5581 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     5641 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     5701 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     5761 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     5821 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
     5881 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5941 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     6001 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     6061 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg ggaagaggcg
     6121 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     6181 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6241 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6301 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6361 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6421 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6481 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6541 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6601 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6661 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6721 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6781 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6841 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6901 gatctcaaga agatcctttg atcttttcta cggggtctga cactcagtgg aacgaaaact
     6961 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     7021 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     7081 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     7141 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7201 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7261 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7321 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7381 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7441 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7501 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7561 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7621 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7681 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7741 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7801 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7861 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7921 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7981 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     8041 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg
     8101 atcccctagg gtcgactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     8161 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     8221 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     8281 tgcttcgcga tgtacgggcc agatatacgc gtt
//
