LOCUS       sequence-379999-        7836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express TadDE (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 TadDE
FEATURES             Location/Qualifiers
     source          1..7836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        6..385
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..589
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     540..560
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     631..650
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        631..649
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             699..719
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4506..4754
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4785..5033
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5076..5096
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5105..5122
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5145..5162)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5151..5375
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5446..5462)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5446..5462)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5459..5481)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5470..5486
                     /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(5494..5500,5501..5518,5519..5524))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5539..5560
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5677..5694)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5848..6436)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5928..5947)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6607..7398,7399..7467))
                     /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     7230..7249
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7468..7572)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7651..7670)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
       61 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      121 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      181 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      241 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      301 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      361 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      421 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      481 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
      541 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa
      601 ccgtcagatc cgctagagat ccgcggccgc taatacgact cactataggg agagccgcca
      661 ccatgaaacg gacagccgac ggaagcgagt tcgagtcacc aaagaagaag cggaaagtca
      721 gttctgaggt ggagttttcc cacgagtact ggatgagaca tgccctgacc ctggccaaga
      781 gggcacggga tgagggagag gcgcctgtgg gagccgtgct ggtgctgaac aatagagtga
      841 tcggcgaggg ctggaacaga cgtatcggcc tgcacgaccc aacagcccat gccgaaatta
      901 tggccctgag acagggcggc ctggtcatgc agaactccag actgattgac gccaccctgt
      961 acgtgacatt cgagccttgc gtgatgtgcg ccggcgccat gatcaactct aggatcggcc
     1021 gcgtggtgtt tggcgtgagg aactcaaaaa gaggcgccgc aggctccctg atgaacgtgc
     1081 tgaactaccc cggcatgaat caccgcgtcg aaattaccga gggaatcctg gcagatgaat
     1141 gtgccgccct gctgtgcgat ttctatcgga tgcctagaca ggtgttcaat gctcagaaga
     1201 aggcccagag ctccatcaac tctggcggat ctagcggagg atcctctggc agcgagacac
     1261 caggaacaag cgagtcagca acaccagaga gcagtggcgg cagcagcggc ggcagcggga
     1321 agcgaaatta cattctgggg ctggccattg gcattacatc agtgggctat ggcatcattg
     1381 actacgagac aagggacgtg atcgacgccg gcgtgagact gttcaaggag gccaacgtgg
     1441 agaacaatga gggccggaga tccaagaggg gagcaaggcg cctgaagcgg agaaggcgcc
     1501 acagaatcca gagagtgaag aagctgctgt tcgattacaa cctgctgacc gaccactccg
     1561 agctgtctgg catcaatcct tatgaggcca gagtgaaggg cctgtcccag aagctgtctg
     1621 aggaggagtt tagcgccgcc ctgctgcacc tggcaaagag gagaggcgtg cacaacgtga
     1681 atgaggtgga ggaggacacc ggcaacgagc tgtccacaaa ggagcagatc agccgcaatt
     1741 ccaaggccct ggaggagaag tatgtggccg agctgcagct ggagcggctg aagaaggatg
     1801 gcgaggtgag gggctccatc aatcgcttca agacctctga ctacgtgaag gaggccaagc
     1861 agctgctgaa ggtgcagaag gcctaccacc agctggatca gtcctttatc gatacatata
     1921 tcgacctgct ggagacaagg cgcacatact atgagggacc aggagagggc tctcccttcg
     1981 gctggaagga catcaaggag tggtacgaga tgctgatggg ccactgcacc tattttccag
     2041 aggagctgag aagcgtgaag tacgcctata acgccgatct gtacaacgcc ctgaatgacc
     2101 tgaacaacct ggtcatcacc agggatgaga acgagaagct ggagtactat gagaagttcc
     2161 agatcatcga gaacgtgttc aagcagaaga agaagcctac actgaagcag atcgccaagg
     2221 agatcctggt gaacgaggag gacatcaagg gctaccgcgt gacctccaca ggcaagccag
     2281 agttcaccaa tctgaaggtg tatcacgata tcaaggacat cacagcccgg aaggagatca
     2341 tcgagaacgc cgagctgctg gatcagatcg ccaagatcct gaccatctat cagagctccg
     2401 aggacatcca ggaggagctg accaacctga atagcgagct gacacaggag gagatcgagc
     2461 agatcagcaa tctgaagggc tacaccggca cacacaacct gagcctgaag gccatcaatc
     2521 tgatcctgga tgagctgtgg cacacaaacg acaatcagat cgccatcttt aaccggctga
     2581 agctggtgcc aaagaaggtg gacctgtccc agcagaagga gatcccaacc acactggtgg
     2641 acgatttcat cctgtctccc gtggtgaagc ggagcttcat ccagagcatc aaagtgatca
     2701 acgccatcat caagaagtac ggcctgccca atgatatcat catcgagctg gccagggaga
     2761 agaactccaa ggacgcccag aagatgatca atgagatgca gaagaggaac cgccagacca
     2821 atgagcggat cgaggagatc atcagaacca caggcaagga gaacgccaag tacctgatcg
     2881 agaagatcaa gctgcacgat atgcaggagg gcaagtgtct gtattctctg gaggccatcc
     2941 ctctggagga cctgctgaac aatccattca actacgaggt ggatcacatc atcccccgga
     3001 gcgtgagctt cgacaattct tttaacaata aggtgctggt gaagcaggag gagaacagca
     3061 agaagggcaa taggacccct ttccagtacc tgtctagctc cgattctaag atcagctacg
     3121 agacattcaa gaagcacatc ctgaatctgg ccaagggcaa gggccgcatc agcaagacca
     3181 agaaggagta cctgctggag gagcgggaca tcaacagatt ctccgtgcag aaggacttca
     3241 tcaaccggaa tctggtggac accagatacg ccacacgcgg cctgatgaat ctgctgcggt
     3301 cttatttcag agtgaacaat ctggatgtga aggtaaagag catcaacggc ggcttcacct
     3361 cctttctgcg gagaaagtgg aagtttaaga aggagcgcaa caagggctat aagcaccacg
     3421 ccgaggatgc cctgatcatc gccaatgccg acttcatctt taaggagtgg aagaagctgg
     3481 acaaggccaa gaaagtgatg gagaaccaga tgttcgagga gaagcaggcc gagagcatgc
     3541 ccgagatcga gacagagcag gagtacaagg agattttcat cacacctcac cagatcaagc
     3601 acatcaagga cttcaaggac tacaagtatt ctcacagggt ggataagaag cccaaccgcg
     3661 agctgatcaa tgacaccctg tatagcacac ggaaggacga taagggcaat accctgatcg
     3721 tgaacaatct gaacggcctg tacgacaagg ataatgacaa gctgaagaag ctgatcaaca
     3781 agtctcccga gaagctgctg atgtaccacc acgatcctca gacatatcag aagctgaagc
     3841 tgatcatgga gcagtacggc gacgagaaga acccactgta taagtactat gaggagacag
     3901 gcaactacct gacaaagtat agcaagaagg ataatggccc cgtgatcaag aagatcaagt
     3961 actatggcaa caagctgaat gcccacctgg acatcaccga cgattaccct aactctcgca
     4021 ataaggtggt gaagctgagc ctgaagccat accggttcga cgtgtacctg gacaacggcg
     4081 tgtataagtt tgtgacagtg aagaatctgg atgtgatcaa gaaggagaac tactatgagg
     4141 tgaacagcaa gtgctacgag gaggccaaga agctgaagaa gatcagcaac caggccgagt
     4201 tcatcgcctc tttttacaac aatgacctga tcaagatcaa tggcgagctg tatagagtga
     4261 tcggcgtgaa caatgatctg ctgaacagaa tcgaagtgaa tatgatcgac atcacctaca
     4321 gggagtatct ggagaacatg aatgataaga ggccccctcg catcatcaag accatcgcct
     4381 ctaagacaca gagcatcaag aagtacagca cagacatcct ggggaacctg tatgaagtca
     4441 agagcaagaa acatcctcag attatcaaga aaggcagcgg cgggagcggc gggagcgggg
     4501 ggagcactaa tctgagcgac atcattgaga aggagactgg gaaacagctg gtcattcagg
     4561 agtccatcct gatgctgcct gaggaggtgg aggaagtgat cggcaacaag ccagagtctg
     4621 acatcctggt gcacaccgcc tacgacgagt ccacagatga gaatgtgatg ctgctgacct
     4681 ctgacgcccc cgagtataag ccttgggccc tggtcatcca ggattctaac ggcgagaata
     4741 agatcaagat gctgagcgga ggatccggag gatctggagg cagcaccaac ctgtctgaca
     4801 tcatcgagaa ggagacaggc aagcagctgg tcatccagga gagcatcctg atgctgcccg
     4861 aagaagtcga agaagtgatc ggaaacaagc ctgagagcga tatcctggtc cataccgcct
     4921 acgacgagag taccgacgaa aatgtgatgc tgctgacatc cgacgcccca gagtataagc
     4981 cctgggctct ggtcatccag gattccaacg gagagaacaa aatcaaaatg ctgtctggcg
     5041 gctcaaaaag aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg aaagtctaac
     5101 cggtcatcat caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc
     5161 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     5221 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     5281 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     5341 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     5401 gggctcgata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5461 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5521 taaagcctag ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5581 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5641 gagaggcggt ttgcgtattg ggcgcacttc cgcttcctcg ctcactgact cgctgcgctc
     5701 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5761 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5821 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5881 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5941 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6001 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6061 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6121 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6181 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6241 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6301 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6361 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6421 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgaca ctcagtggaa
     6481 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6541 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6601 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6661 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6721 tggccccagt gctgcaatga taccgcggga cccacgctca ccggctccag atttatcagc
     6781 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6841 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6901 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6961 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7021 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7081 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7141 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7201 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7261 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7321 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7381 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7441 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7501 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7561 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcg
     7621 atctcccgat cccctagggt cgactctcag tacaatctgc tctgatgccg catagttaag
     7681 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     7741 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     7801 ttttgcgctg cttcgcgatg tacgggccag atatac
//
