LOCUS       sequence-379995-        7836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express TadCBEa (with SaCas9) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7836)
  AUTHORS   Neugebauer ME, Hsu A, Arbab M, Krasnow NA, McElroy AN, Pandey S, 
            Doman JL, Huang TP, Raguram A, Banskota S, Newby GA, Tolar J, Osborn
            MJ, Liu DR
  TITLE     Evolution of an adenine base editor into a small, efficient cytosine
            base editor with low off-target activity.
  JOURNAL   Nat Biotechnol. 2022 Nov 10. pii: 10.1038/s41587-022-01533-6. doi: 
            10.1038/s41587-022-01533-6.
  PUBMED    36357719
REFERENCE   2  (bases 1 to 7836)
  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. 2022 Nov 10. pii: 10.1038/s41587-022-01533-6. doi: 
            10.1038/s41587-022-01533-6."
COMMENT     Sequence Label: SaCas9 TadCBEa
FEATURES             Location/Qualifiers
     source          1..7836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        377..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     531..551
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     622..641
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        622..640
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             690..710
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4497..4745
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4776..5024
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5067..5087
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5096..5113
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5136..5153)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5142..5366
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5437..5453)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5437..5453)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5450..5472)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5461..5477
                     /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(5485..5491,5492..5509,5510..5515))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5530..5551
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5668..5685)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5839..6427)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5919..5938)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6598..7389,7390..7458))
                     /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     7221..7240
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7459..7563)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7642..7661)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7833..7836,1..376)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
       61 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      121 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      181 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      241 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      301 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      361 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      421 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      481 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      541 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
      601 ccgctagaga tccgcggccg ctaatacgac tcactatagg gagagccgcc accatgaaac
      661 ggacagccga cggaagcgag ttcgagtcac caaagaagaa gcggaaagtc agttctgagg
      721 tggagttttc ccacgagtac tggatgagac atgccctgac cctggccaag agggcacggg
      781 atgagggggc gggtcctgtg ggagccgtgc tggtgctgaa caatagagtg atcggcgagg
      841 gctggaacag agccatcggc ctgcacgacc caacagccca tgccgaaatt atggccctga
      901 gacagggcgg cctggtcatg cagaactaca gactgtttga cgccaccctg tacgtgacat
      961 tcgagccttg cgtgatgtgc gccggcgcca tgatcaactc taggatcggc cgcgtggtgt
     1021 ttggcgtgag gaactcaaaa agaggcgccg caggctccct gatgaacgtg ctgaactacc
     1081 ccggcatgaa tcaccgcgtc gaaattaccg agggaatcct ggcagatgaa tgtgccgccc
     1141 tgctgtgcga tttctatcgg atgcctagac aggtgttcaa ttctcagaag aaggcccaga
     1201 gctccatcaa ctctggcgga tctagcggag gatcctctgg cagcgagaca ccaggaacaa
     1261 gcgagtcagc aacaccagag agcagtggcg gcagcagcgg cggcagcggg aagcgaaatt
     1321 acattctggg gctggccatt ggcattacat cagtgggcta tggcatcatt gactacgaga
     1381 caagggacgt gatcgacgcc ggcgtgagac tgttcaagga ggccaacgtg gagaacaatg
     1441 agggccggag atccaagagg ggagcaaggc gcctgaagcg gagaaggcgc cacagaatcc
     1501 agagagtgaa gaagctgctg ttcgattaca acctgctgac cgaccactcc gagctgtctg
     1561 gcatcaatcc ttatgaggcc agagtgaagg gcctgtccca gaagctgtct gaggaggagt
     1621 ttagcgccgc cctgctgcac ctggcaaaga ggagaggcgt gcacaacgtg aatgaggtgg
     1681 aggaggacac cggcaacgag ctgtccacaa aggagcagat cagccgcaat tccaaggccc
     1741 tggaggagaa gtatgtggcc gagctgcagc tggagcggct gaagaaggat ggcgaggtga
     1801 ggggctccat caatcgcttc aagacctctg actacgtgaa ggaggccaag cagctgctga
     1861 aggtgcagaa ggcctaccac cagctggatc agtcctttat cgatacatat atcgacctgc
     1921 tggagacaag gcgcacatac tatgagggac caggagaggg ctctcccttc ggctggaagg
     1981 acatcaagga gtggtacgag atgctgatgg gccactgcac ctattttcca gaggagctga
     2041 gaagcgtgaa gtacgcctat aacgccgatc tgtacaacgc cctgaatgac ctgaacaacc
     2101 tggtcatcac cagggatgag aacgagaagc tggagtacta tgagaagttc cagatcatcg
     2161 agaacgtgtt caagcagaag aagaagccta cactgaagca gatcgccaag gagatcctgg
     2221 tgaacgagga ggacatcaag ggctaccgcg tgacctccac aggcaagcca gagttcacca
     2281 atctgaaggt gtatcacgat atcaaggaca tcacagcccg gaaggagatc atcgagaacg
     2341 ccgagctgct ggatcagatc gccaagatcc tgaccatcta tcagagctcc gaggacatcc
     2401 aggaggagct gaccaacctg aatagcgagc tgacacagga ggagatcgag cagatcagca
     2461 atctgaaggg ctacaccggc acacacaacc tgagcctgaa ggccatcaat ctgatcctgg
     2521 atgagctgtg gcacacaaac gacaatcaga tcgccatctt taaccggctg aagctggtgc
     2581 caaagaaggt ggacctgtcc cagcagaagg agatcccaac cacactggtg gacgatttca
     2641 tcctgtctcc cgtggtgaag cggagcttca tccagagcat caaagtgatc aacgccatca
     2701 tcaagaagta cggcctgccc aatgatatca tcatcgagct ggccagggag aagaactcca
     2761 aggacgccca gaagatgatc aatgagatgc agaagaggaa ccgccagacc aatgagcgga
     2821 tcgaggagat catcagaacc acaggcaagg agaacgccaa gtacctgatc gagaagatca
     2881 agctgcacga tatgcaggag ggcaagtgtc tgtattctct ggaggccatc cctctggagg
     2941 acctgctgaa caatccattc aactacgagg tggatcacat catcccccgg agcgtgagct
     3001 tcgacaattc ttttaacaat aaggtgctgg tgaagcagga ggagaacagc aagaagggca
     3061 ataggacccc tttccagtac ctgtctagct ccgattctaa gatcagctac gagacattca
     3121 agaagcacat cctgaatctg gccaagggca agggccgcat cagcaagacc aagaaggagt
     3181 acctgctgga ggagcgggac atcaacagat tctccgtgca gaaggacttc atcaaccgga
     3241 atctggtgga caccagatac gccacacgcg gcctgatgaa tctgctgcgg tcttatttca
     3301 gagtgaacaa tctggatgtg aaggtaaaga gcatcaacgg cggcttcacc tcctttctgc
     3361 ggagaaagtg gaagtttaag aaggagcgca acaagggcta taagcaccac gccgaggatg
     3421 ccctgatcat cgccaatgcc gacttcatct ttaaggagtg gaagaagctg gacaaggcca
     3481 agaaagtgat ggagaaccag atgttcgagg agaagcaggc cgagagcatg cccgagatcg
     3541 agacagagca ggagtacaag gagattttca tcacacctca ccagatcaag cacatcaagg
     3601 acttcaagga ctacaagtat tctcacaggg tggataagaa gcccaaccgc gagctgatca
     3661 atgacaccct gtatagcaca cggaaggacg ataagggcaa taccctgatc gtgaacaatc
     3721 tgaacggcct gtacgacaag gataatgaca agctgaagaa gctgatcaac aagtctcccg
     3781 agaagctgct gatgtaccac cacgatcctc agacatatca gaagctgaag ctgatcatgg
     3841 agcagtacgg cgacgagaag aacccactgt ataagtacta tgaggagaca ggcaactacc
     3901 tgacaaagta tagcaagaag gataatggcc ccgtgatcaa gaagatcaag tactatggca
     3961 acaagctgaa tgcccacctg gacatcaccg acgattaccc taactctcgc aataaggtgg
     4021 tgaagctgag cctgaagcca taccggttcg acgtgtacct ggacaacggc gtgtataagt
     4081 ttgtgacagt gaagaatctg gatgtgatca agaaggagaa ctactatgag gtgaacagca
     4141 agtgctacga ggaggccaag aagctgaaga agatcagcaa ccaggccgag ttcatcgcct
     4201 ctttttacaa caatgacctg atcaagatca atggcgagct gtatagagtg atcggcgtga
     4261 acaatgatct gctgaacaga atcgaagtga atatgatcga catcacctac agggagtatc
     4321 tggagaacat gaatgataag aggccccctc gcatcatcaa gaccatcgcc tctaagacac
     4381 agagcatcaa gaagtacagc acagacatcc tggggaacct gtatgaagtc aagagcaaga
     4441 aacatcctca gattatcaag aaaggcagcg gcgggagcgg cgggagcggg gggagcacta
     4501 atctgagcga catcattgag aaggagactg ggaaacagct ggtcattcag gagtccatcc
     4561 tgatgctgcc tgaggaggtg gaggaagtga tcggcaacaa gccagagtct gacatcctgg
     4621 tgcacaccgc ctacgacgag tccacagatg agaatgtgat gctgctgacc tctgacgccc
     4681 ccgagtataa gccttgggcc ctggtcatcc aggattctaa cggcgagaat aagatcaaga
     4741 tgctgagcgg aggatccgga ggatctggag gcagcaccaa cctgtctgac atcatcgaga
     4801 aggagacagg caagcagctg gtcatccagg agagcatcct gatgctgccc gaagaagtcg
     4861 aagaagtgat cggaaacaag cctgagagcg atatcctggt ccataccgcc tacgacgaga
     4921 gtaccgacga aaatgtgatg ctgctgacat ccgacgcccc agagtataag ccctgggctc
     4981 tggtcatcca ggattccaac ggagagaaca aaatcaaaat gctgtctggc ggctcaaaaa
     5041 gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtctaa ccggtcatca
     5101 tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     5161 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     5221 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     5281 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     5341 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgat
     5401 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5461 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagccta
     5521 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5581 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5641 tttgcgtatt gggcgcactt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5701 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5761 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5821 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5881 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5941 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6001 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6061 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6121 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6181 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6241 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6301 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6361 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6421 atctcaagaa gatcctttga tcttttctac ggggtctgac actcagtgga acgaaaactc
     6481 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6541 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6601 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6661 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6721 tgctgcaatg ataccgcggg acccacgctc accggctcca gatttatcag caataaacca
     6781 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6841 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6901 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6961 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7021 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7081 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7141 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7201 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7261 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7321 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7381 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7441 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7501 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7561 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc gatctcccga
     7621 tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     7681 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     7741 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     7801 gcttcgcgat gtacgggcca gatatacgcg ttgaca
//
