LOCUS       sequence-424823-        7917 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express CBE6a (with eNme2-C variant) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7917)
  AUTHORS   Zhang E, Neugebauer ME, Krasnow NA, Liu DR
  TITLE     Phage-assisted evolution of highly active cytosine base editors with
            enhanced selectivity and minimal sequence context preference.
  JOURNAL   Nat Commun. 2024 Feb 24;15(1):1697. doi: 10.1038/s41467-024-45969-7.
  PUBMED    38402281
REFERENCE   2  (bases 1 to 7917)
  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 
            Commun."; date: "2024-02-24"; volume: "15(1):1697. doi"; pages: " 
            10.1038/s41467-024-45969-7"
COMMENT     Sequence Label: eNme2C-CBE6a
FEATURES             Location/Qualifiers
     source          1..7917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     610..629
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        610..628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             678..698
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4566..4814
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4845..5093
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5136..5156
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5165..5182
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5205..5222)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5211..5435
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5506..5522)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5506..5522)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5519..5541)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5530..5546
                     /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(5554..5560,5561..5578,5579..5584))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5599..5620
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5737..5754)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5908..6496)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5988..6007)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6667..7458,7459..7527))
                     /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     7290..7309
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7528..7632)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7711..7730)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7902..7917,1..364)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc
      601 cgcggccgct aatacgactc actataggga gagccgccac catgaaacgg acagccgacg
      661 gaagcgagtt cgagtcacca aagaagaagc ggaaagtcag ttctgaggtg gagttttccc
      721 acgagtactg gatgagacat gccctgaccc tggccaagag ggcacgggat gagggagagg
      781 cgcctgtggg agccgtgctg gtgctgaaca atagagtgat cggcgagggc tggatcagac
      841 gtatcggcct gcacgaccca acagcccatg ccgaaattat ggccctgaga cagggcggcc
      901 tggtcatgca gaaccccaga ctgattgacg ccaccctgta cgtgacattc gagccttgcg
      961 tgatgtgcgc cggcgccatg atcaactcta ggatcggccg cgtggtgttt ggcgtgagga
     1021 actcaaaaag aggcgccgca ggctccctga tgaacgtgct gaactacccc ggcatgaatc
     1081 accgcgtcga aattaccgag ggaatcctgg cagatgaatg tgccgccctg ctgtgcgatt
     1141 tctatcggat gcctagacag gtgttcaatg ctcagaagaa ggcccagagc tccatcaact
     1201 ctggcggatc tagcggagga tcctctggca gcgagacacc aggaacaagc gagtcagcaa
     1261 caccagagag cagtggcggc agcagcggcg gcagcgcagc attcaagcct aacccaatca
     1321 attacatcct gggactggca atcggaatcg catccgtggg atgggctatg gtggagatcg
     1381 acgaggaggg gaatcctatc cggctgatcg atctgggcgt gagagtgttt gagagggccg
     1441 aggtgccaaa gaccggcgat tctctggcta tggcccggag actggcacgg agcgtgaggc
     1501 gcctgacacg gagaagggca cacaggctgc tgagggcacg ccggctgctg aagagagagg
     1561 gcgtgctgca ggcagcagac ttcgatgaga atggcctgat cacgagcttg ccaaacaccc
     1621 cctggcagct gagagcagcc gccctggaca ggaagctgac accactggag tggtctgccg
     1681 tgctgctgca cctgatcaag caccgcggct acctgagcca gcggaagaac gagggagaga
     1741 cagcagccaa ggagctgggc gccctgctga agggagtggc caacaatgcc cacgccctgc
     1801 agaccggcga tttcaggaca cctgccgagc tggccctgaa taagtttgag aaggagtccg
     1861 gccacatcag aaaccagagg ggcgactata gccacacctt ctcccgcaag gatctgcagg
     1921 ccgagctgat cctgctgttc gagaagcaga aggagtttgg caatccacac gtgagcggag
     1981 gcctgaagga gggaatcgag accctgctga tgacacagag gcctgccctg tccggcgacg
     2041 cagtgcagaa gatgctgggg cactgcaccc tcgagcctac agagccaaag gccgccaaga
     2101 acacctacac agccgagcgg tttatctggc tgacaaagct gaacaatctg agaatcctgg
     2161 agcagggatc cgagaggcca ctgaccgaca cagagaggtc caccctgatg gatgagcctt
     2221 accggaagtc taaactgaca tatgcccagg ccagaaagct gctgggcctg gaggacaccg
     2281 ccttctttaa gggcctgaga tacggcaagg ataatgccga ggcctccaca ctgatggaga
     2341 tgaaggccta tcacgccatc tctcgcgccc tggagaagga gggcctgaag gacaagaagt
     2401 cccccctgaa cctgagctcc gagctgcagg atgagatcgg caccgccttc tctctgttta
     2461 agaccgacga ggatatcaca ggccgcctga aggacagggt gcagcctgag atcctggagg
     2521 ccctgctgaa gcacatctct ttcgataagt ttgtgcagat cagcctgaag gccctgagaa
     2581 ggatcgtgcc actgatggag cagggcaagc ggtacgacga ggcctgcgcc gagatctacg
     2641 gcgttcacta tggcaagaag aacacagagg agaagatcta tctgccccct atccctgccg
     2701 acgagatcag aaatcctgtg gtgctgaggg ccctgtccca ggcaagaaaa gtgatcaacg
     2761 gagtggtgcg ccggtacgga tctccagccc ggatccacat cgagaccgcc agagaagtgg
     2821 gcaagagctt caaggaccgg aaggagatcg cgaagagaca ggaggagaat cgcaaggatc
     2881 gggagaaggc cgccgccaag tttagggagt acttccctaa ctttgtgggc gagccaaagt
     2941 ctaaggacat cctgaagctg cgcctgtacg agcagcagca cggcaagtgt ctgtatagcg
     3001 gcaaagagat caatctggtg cggctgaacg agaagggcta tgtggagatc gatcacgccc
     3061 tgcctttctc cagaacctgg gacgattctt ttaacaataa ggtgctggtg ctgggcagcg
     3121 agaaccagaa taagggcaat cagacaccat acgagtattt caatggcaag gacaactcca
     3181 gggagtggca ggagttcaag gcccgcgtgg agacctctag atttcccagt agcaagaagc
     3241 agcggatcct gctgcagaag ttcgacgagg atggctttaa ggagtgcaac ctgaatgaca
     3301 ccagatacgt gaaccggttc ctgtgccagt ttgtggccga tcacatcctg ctgaccggca
     3361 agggcaagag aagggtggtc gcctctaatg gccagatcac aaacctgctg agggggtttt
     3421 ggagactgag gaaggtgcgg gcagagaatg acagacacca cgcactggat gcagtggtgg
     3481 tggcatgcag caccgtggca atgcagcaga agatcacaag attcgtgagg tataaggaga
     3541 tgaacgcctt tgacggcaag accgtcgata aggagacagg caaggtgctg taccagaaga
     3601 cccacttccc ccagccttgg gagttctttg cccaggaagt tatgatccgg gtgttcggca
     3661 agccagacgg caagcctgag tttgaggagg ccgatacccc agagaagctg aggacactgc
     3721 tggcagagaa gctgtctagc aggccagagg cagtgcacga gtacgtgacc ccgctgttcg
     3781 tgtccagggc acccaatcgg aagatgtctg gcgcccacaa ggacacactg agaagcgcca
     3841 agaggtttgt gaagcacaac gagaagatct ccgtgaagag agtgtggctg accgagatca
     3901 agctggccga tctggagaac atggtgaatt acaagaacgg cagggagatc gagctgtatg
     3961 aggccctgaa ggcaaggctg gaggcctacg gaggaaatgc caagcaggcc ttcgacccaa
     4021 aggataaccc cttttataag aagggaggac agctggtgaa ggccgtgcgg gtggagaaga
     4081 cccagaaaag cggcgtgctg ctgaataaga agaacgccta cacaatcgcc gacaatggtg
     4141 atatggtgag agtggacgtg ttctgtaagg tggataagaa gggcaagaat cagtacttta
     4201 tcgtgcctat ctatgcctgg caggtggccg agaacatcct gccagacatc gattgcaagg
     4261 gctacagaat cgacgatagc tatacattct gtttttccct gcacaagtat gacctgatcg
     4321 ccttccagaa ggatgagaag tccaaggtgg agtttgccta ctatatcaat tgcgactcct
     4381 ctagcggcgg gttctacctg gcctggcacg ataagggcag cagggagcag cggtttcgca
     4441 tctccaccca gaatctggcg ctgatccaga agtatcaggt gaacgagctg ggcaaggaga
     4501 tcaggccatg tcggctgaag aagcgcccac ccgtgagcgg cgggagcggc gggagcgggg
     4561 ggagcactaa tctgagcgac atcattgaga aggagactgg gaaacagctg gtcattcagg
     4621 agtccatcct gatgctgcct gaggaggtgg aggaagtgat cggcaacaag ccagagtctg
     4681 acatcctggt gcacaccgcc tacgacgagt ccacagatga gaatgtgatg ctgctgacct
     4741 ctgacgcccc cgagtataag ccttgggccc tggtcatcca ggattctaac ggcgagaata
     4801 agatcaagat gctgagcgga ggatccggag gatctggagg cagcaccaac ctgtctgaca
     4861 tcatcgagaa ggagacaggc aagcagctgg tcatccagga gagcatcctg atgctgcccg
     4921 aagaagtcga agaagtgatc ggaaacaagc ctgagagcga tatcctggtc cataccgcct
     4981 acgacgagag taccgacgaa aatgtgatgc tgctgacatc cgacgcccca gagtataagc
     5041 cctgggctct ggtcatccag gattccaacg gagagaacaa aatcaaaatg ctgtctggcg
     5101 gctcaaaaag aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg aaagtctaac
     5161 cggtcatcat caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc
     5221 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     5281 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     5341 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     5401 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     5461 gggctcgata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5521 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5581 taaagcctag ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5641 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5701 gagaggcggt ttgcgtattg ggcgcacttc cgcttcctcg ctcactgact cgctgcgctc
     5761 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5821 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5881 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5941 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6001 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6061 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6121 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6181 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6241 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6301 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6361 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6421 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6481 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgaca ctcagtggaa
     6541 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6601 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6661 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6721 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6781 tggccccagt gctgcaatga taccgcggga cccacgctca ccggctccag atttatcagc
     6841 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6901 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6961 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7021 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7081 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7141 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7201 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7261 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7321 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7381 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7441 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7501 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7561 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7621 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcg
     7681 atctcccgat cccctagggt cgactctcag tacaatctgc tctgatgccg catagttaag
     7741 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     7801 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     7861 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgac
//
