LOCUS       sequence-379997-        7836 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express TadCBEd (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 Apr 14, 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 TadCBEd
FEATURES             Location/Qualifiers
     source          1..7836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        378..581
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     532..552
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             691..711
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4498..4746
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4777..5025
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5068..5088
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5097..5114
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5137..5154)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5143..5367
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5438..5454)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5438..5454)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5451..5473)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5462..5478
                     /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(5486..5492,5493..5510,5511..5516))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5531..5552
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5669..5686)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5840..6428)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5920..5939)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6599..7390,7391..7459))
                     /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     7222..7241
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7460..7564)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7643..7662)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7834..7836,1..377)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
       61 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      121 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      181 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      241 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      301 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      361 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      421 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      481 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      541 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga
      601 tccgctagag atccgcggcc gctaatacga ctcactatag ggagagccgc caccatgaaa
      661 cggacagccg acggaagcga gttcgagtca ccaaagaaga agcggaaagt cagttctgag
      721 gtggagtttt cccacgagta ctggatgaga catgccctga ccctggccaa gagggcacgg
      781 gatgagagga aggcgcctgt gggagccgtg ctggtgctga acaatagagt gatcggcgag
      841 ggctggaaca gagccatcgg cctgcacgac ccaacagccc atgccgaaat tatagccctg
      901 agacagggcg gcctggtcat gcagaactac agactgattg acgccaccct gtacgtgaca
      961 ttcgagcctt gcgtgatgtg cgccggcgcc atgatcaact ctaggatcgg ccgcgtggtg
     1021 tttggcgtga ggaactcaaa aagaggcgcc gcaggctccc tgatgaacgt gctgaactac
     1081 cccggaatga atcaccgcgt cgaaattacc gagggaatcc tggcagatga atgtgccgcc
     1141 ctgctgtgcg atttctatcg gatgcctaga caggtgttca atgctcagaa gaaggcccag
     1201 agctccatca actctggcgg atctagcgga ggatcctctg gcagcgagac accaggaaca
     1261 agcgagtcag caacaccaga gagcagtggc ggcagcagcg gcggcagcgg gaagcgaaat
     1321 tacattctgg ggctggccat tggcattaca tcagtgggct atggcatcat tgactacgag
     1381 acaagggacg tgatcgacgc cggcgtgaga ctgttcaagg aggccaacgt ggagaacaat
     1441 gagggccgga gatccaagag gggagcaagg cgcctgaagc ggagaaggcg ccacagaatc
     1501 cagagagtga agaagctgct gttcgattac aacctgctga ccgaccactc cgagctgtct
     1561 ggcatcaatc cttatgaggc cagagtgaag ggcctgtccc agaagctgtc tgaggaggag
     1621 tttagcgccg ccctgctgca cctggcaaag aggagaggcg tgcacaacgt gaatgaggtg
     1681 gaggaggaca ccggcaacga gctgtccaca aaggagcaga tcagccgcaa ttccaaggcc
     1741 ctggaggaga agtatgtggc cgagctgcag ctggagcggc tgaagaagga tggcgaggtg
     1801 aggggctcca tcaatcgctt caagacctct gactacgtga aggaggccaa gcagctgctg
     1861 aaggtgcaga aggcctacca ccagctggat cagtccttta tcgatacata tatcgacctg
     1921 ctggagacaa ggcgcacata ctatgaggga ccaggagagg gctctccctt cggctggaag
     1981 gacatcaagg agtggtacga gatgctgatg ggccactgca cctattttcc agaggagctg
     2041 agaagcgtga agtacgccta taacgccgat ctgtacaacg ccctgaatga cctgaacaac
     2101 ctggtcatca ccagggatga gaacgagaag ctggagtact atgagaagtt ccagatcatc
     2161 gagaacgtgt tcaagcagaa gaagaagcct acactgaagc agatcgccaa ggagatcctg
     2221 gtgaacgagg aggacatcaa gggctaccgc gtgacctcca caggcaagcc agagttcacc
     2281 aatctgaagg tgtatcacga tatcaaggac atcacagccc ggaaggagat catcgagaac
     2341 gccgagctgc tggatcagat cgccaagatc ctgaccatct atcagagctc cgaggacatc
     2401 caggaggagc tgaccaacct gaatagcgag ctgacacagg aggagatcga gcagatcagc
     2461 aatctgaagg gctacaccgg cacacacaac ctgagcctga aggccatcaa tctgatcctg
     2521 gatgagctgt ggcacacaaa cgacaatcag atcgccatct ttaaccggct gaagctggtg
     2581 ccaaagaagg tggacctgtc ccagcagaag gagatcccaa ccacactggt ggacgatttc
     2641 atcctgtctc ccgtggtgaa gcggagcttc atccagagca tcaaagtgat caacgccatc
     2701 atcaagaagt acggcctgcc caatgatatc atcatcgagc tggccaggga gaagaactcc
     2761 aaggacgccc agaagatgat caatgagatg cagaagagga accgccagac caatgagcgg
     2821 atcgaggaga tcatcagaac cacaggcaag gagaacgcca agtacctgat cgagaagatc
     2881 aagctgcacg atatgcagga gggcaagtgt ctgtattctc tggaggccat ccctctggag
     2941 gacctgctga acaatccatt caactacgag gtggatcaca tcatcccccg gagcgtgagc
     3001 ttcgacaatt cttttaacaa taaggtgctg gtgaagcagg aggagaacag caagaagggc
     3061 aataggaccc ctttccagta cctgtctagc tccgattcta agatcagcta cgagacattc
     3121 aagaagcaca tcctgaatct ggccaagggc aagggccgca tcagcaagac caagaaggag
     3181 tacctgctgg aggagcggga catcaacaga ttctccgtgc agaaggactt catcaaccgg
     3241 aatctggtgg acaccagata cgccacacgc ggcctgatga atctgctgcg gtcttatttc
     3301 agagtgaaca atctggatgt gaaggtaaag agcatcaacg gcggcttcac ctcctttctg
     3361 cggagaaagt ggaagtttaa gaaggagcgc aacaagggct ataagcacca cgccgaggat
     3421 gccctgatca tcgccaatgc cgacttcatc tttaaggagt ggaagaagct ggacaaggcc
     3481 aagaaagtga tggagaacca gatgttcgag gagaagcagg ccgagagcat gcccgagatc
     3541 gagacagagc aggagtacaa ggagattttc atcacacctc accagatcaa gcacatcaag
     3601 gacttcaagg actacaagta ttctcacagg gtggataaga agcccaaccg cgagctgatc
     3661 aatgacaccc tgtatagcac acggaaggac gataagggca ataccctgat cgtgaacaat
     3721 ctgaacggcc tgtacgacaa ggataatgac aagctgaaga agctgatcaa caagtctccc
     3781 gagaagctgc tgatgtacca ccacgatcct cagacatatc agaagctgaa gctgatcatg
     3841 gagcagtacg gcgacgagaa gaacccactg tataagtact atgaggagac aggcaactac
     3901 ctgacaaagt atagcaagaa ggataatggc cccgtgatca agaagatcaa gtactatggc
     3961 aacaagctga atgcccacct ggacatcacc gacgattacc ctaactctcg caataaggtg
     4021 gtgaagctga gcctgaagcc ataccggttc gacgtgtacc tggacaacgg cgtgtataag
     4081 tttgtgacag tgaagaatct ggatgtgatc aagaaggaga actactatga ggtgaacagc
     4141 aagtgctacg aggaggccaa gaagctgaag aagatcagca accaggccga gttcatcgcc
     4201 tctttttaca acaatgacct gatcaagatc aatggcgagc tgtatagagt gatcggcgtg
     4261 aacaatgatc tgctgaacag aatcgaagtg aatatgatcg acatcaccta cagggagtat
     4321 ctggagaaca tgaatgataa gaggccccct cgcatcatca agaccatcgc ctctaagaca
     4381 cagagcatca agaagtacag cacagacatc ctggggaacc tgtatgaagt caagagcaag
     4441 aaacatcctc agattatcaa gaaaggcagc ggcgggagcg gcgggagcgg ggggagcact
     4501 aatctgagcg acatcattga gaaggagact gggaaacagc tggtcattca ggagtccatc
     4561 ctgatgctgc ctgaggaggt ggaggaagtg atcggcaaca agccagagtc tgacatcctg
     4621 gtgcacaccg cctacgacga gtccacagat gagaatgtga tgctgctgac ctctgacgcc
     4681 cccgagtata agccttgggc cctggtcatc caggattcta acggcgagaa taagatcaag
     4741 atgctgagcg gaggatccgg aggatctgga ggcagcacca acctgtctga catcatcgag
     4801 aaggagacag gcaagcagct ggtcatccag gagagcatcc tgatgctgcc cgaagaagtc
     4861 gaagaagtga tcggaaacaa gcctgagagc gatatcctgg tccataccgc ctacgacgag
     4921 agtaccgacg aaaatgtgat gctgctgaca tccgacgccc cagagtataa gccctgggct
     4981 ctggtcatcc aggattccaa cggagagaac aaaatcaaaa tgctgtctgg cggctcaaaa
     5041 agaaccgccg acggcagcga attcgagccc aagaagaaga ggaaagtcta accggtcatc
     5101 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     5161 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     5221 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     5281 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     5341 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
     5401 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5461 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5521 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5581 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5641 gtttgcgtat tgggcgcact tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5701 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5761 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5821 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5881 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5941 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6001 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6061 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6121 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6181 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6241 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6301 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6361 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6421 gatctcaaga agatcctttg atcttttcta cggggtctga cactcagtgg aacgaaaact
     6481 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6541 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6601 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6661 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6721 gtgctgcaat gataccgcgg gacccacgct caccggctcc agatttatca gcaataaacc
     6781 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6841 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6901 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6961 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7021 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7081 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7141 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7201 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7261 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7321 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7381 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7441 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7501 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7561 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg
     7621 atcccctagg gtcgactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     7681 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     7741 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     7801 tgcttcgcga tgtacgggcc agatatacgc gttgac
//
