LOCUS       sequence-307641-        7185 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses split DddAtox-Cas9 construct in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7185)
  AUTHORS   Mok BY, de Moraes MH, Zeng J, Bosch DE, Kotrys AV, Raguram A, Hsu F,
            Radey MC, Peterson SB, Mootha VK, Mougous JD, Liu DR
  TITLE     A bacterial cytidine deaminase toxin enables CRISPR-free 
            mitochondrial base editing.
  JOURNAL   Nature. 2020 Jul 8. pii: 10.1038/s41586-020-2477-4. doi: 
            10.1038/s41586-020-2477-4.
  PUBMED    32641830
REFERENCE   2  (bases 1 to 7185)
  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: "Nature. 
            2020 Jul 8. pii: 10.1038/s41586-020-2477-4. doi: 
            10.1038/s41586-020-2477-4."
COMMENT     Sequence Label: G1333 DddAtox-N–SaKKH-Cas9(D10A)
FEATURES             Location/Qualifiers
     source          1..7185
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      403..412
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3805..3825
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3832..3897
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3910..4158
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4171..4191
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4200..4217
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4240..4257)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4246..4470
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4541..4557)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4541..4557)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4554..4576)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4565..4581
                     /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(4589..4595,4596..4613,4614..4619))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4634..4655
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4772..4789)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4943..5531)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5023..5042)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5702..6493,6494..6562))
                     /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     6325..6344
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6563..6667)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6746..6765)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6937..7185,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gggcagctac
      421 gccctgggtc cgtatcagat tagcgccccg cagctgccag cttacaatgg tcagaccgtg
      481 ggtaccttct actatgtgaa cgacgcgggc ggtctggaga gcaaggtgtt tagcagcggc
      541 ggttctggtg gttcttctgg tggttctagc ggcagcgaga ctcccgggac ctcagagtcc
      601 gccacacccg aaagtagtgg cggcagcagc ggcggcagcg ggaaacggaa ctacatcctg
      661 gggcttgcca ttgggataac cagcgttggc tacggaatta ttgattatga gacacgcgat
      721 gtgattgacg ccggggttag gctgttcaaa gaggccaacg ttgaaaacaa cgagggaaga
      781 cggagtaagc gcggagcaag aagactcaag cgcagacgga gacatcggat tcagagggtg
      841 aaaaagctgc tcttcgatta caatctcctg accgatcata gtgagctgag cggaatcaac
      901 ccctacgagg cgcgagtgaa agggctttcc cagaagctgt ccgaagagga gttctccgcc
      961 gcgttgctgc acctggccaa acggaggggg gttcacaatg taaacgaagt ggaggaggac
     1021 acgggcaatg aacttagtac gaaagaacag atcagtagga actctaaggc tctcgaagag
     1081 aaatacgtcg ctgagttgca gcttgagaga ctgaaaaaag acggcgaagt acgcggatct
     1141 attaataggt tcaagacttc agattacgta aaggaagcca agcagctcct gaaagtacag
     1201 aaagcgtacc atcagctcga tcagagcttc atcgatacct acatagattt gctggagaca
     1261 cggaggacat actacgaggg cccaggggaa ggatctcctt ttgggtggaa ggacatcaag
     1321 gaatggtacg agatgcttat gggacattgt acatattttc cggaggagct caggagcgtc
     1381 aagtacgcct acaatgccga cctgtacaat gccctcaatg acctcaataa cctcgtgatt
     1441 accagggacg agaacgagaa gctggagtac tatgaaaagt tccagattat cgagaatgtg
     1501 tttaagcaga agaagaagcc gacacttaag cagattgcaa aggaaatcct cgtgaatgag
     1561 gaagatatca agggatacag agtgacaagt acaggcaagc ccgagttcac aaatctgaag
     1621 gtgtaccacg atattaagga cataaccgca cgaaaggaga taatcgaaaa cgctgagctc
     1681 ctcgatcaga tcgcaaaaat tcttaccatc taccagtcta gtgaggacat tcaggaggaa
     1741 ctgactaatc tgaacagtga gctcacccaa gaggaaattg agcagatttc aaacctgaaa
     1801 ggctacaccg ggacgcacaa tctgagcctc aaagcaatca acctcattct ggatgaactt
     1861 tggcacacaa atgacaacca aattgccata ttcaaccgcc tgaaactggt gccaaaaaaa
     1921 gtggatctgt cacagcaaaa ggaaatccct acaaccttgg ttgacgattt tattctgtcc
     1981 cccgttgtca agcggagctt catccagtca atcaaggtga tcaatgccat cattaaaaaa
     2041 tacggattgc caaacgatat aattatcgag cttgcacgag agaagaactc aaaggacgcc
     2101 cagaagatga ttaacgaaat gcagaagcgc aaccgccaga caaacgaacg catagaggaa
     2161 attataagaa caaccggcaa agagaatgcc aagtatctga tcgagaaaat caagctgcac
     2221 gacatgcaag aaggcaagtg cctgtactct ctggaagcta tcccactcga agatctgctg
     2281 aataatccat tcaattacga ggtggaccac atcatcccta gatccgtaag ctttgacaat
     2341 tccttcaata acaaagttct ggttaaacag gaggaaaatt ctaaaaaagg gaaccggacc
     2401 ccgttccagt acctgagctc cagtgacagc aagattagct acgagacttt taagaaacat
     2461 attctgaatc tggccaaagg caaaggcagg atcagcaaga ccaagaagga gtacctcctc
     2521 gaagaacgcg acattaacag atttagtgtg cagaaagatt tcatcaaccg aaaccttgtc
     2581 gatactcggt acgccacgag aggcctgatg aatctcctca ggagctactt ccgcgtcaat
     2641 aatctggacg ttaaagtcaa gagcataaat gggggattca ccagctttct gaggagaaag
     2701 tggaagttta agaaggaacg aaacaaagga tacaagcacc atgctgagga tgctttgatc
     2761 atcgctaacg cggactttat ctttaaggaa tggaaaaagc tggataaggc aaagaaagtg
     2821 atggaaaacc agatgttcga ggagaagcag gcagagtcaa tgcctgagat cgagacagag
     2881 caggaataca aggaaatttt catcacccct catcagatta aacacataaa ggacttcaaa
     2941 gactataaat actctcatag ggtggacaaa aaacccaatc gcaagctcat taatgacacc
     3001 ctgtactcaa cacggaagga tgataaaggt aataccttga ttgtgaataa tcttaatgga
     3061 ttgtatgaca aagataacga caagctcaag aagctgatca acaagtctcc agagaagctc
     3121 cttatgtatc accacgaccc acagacttat cagaaattga aactgatcat ggagcaatac
     3181 ggggatgaga agaacccact ctacaaatat tatgaggaaa caggtaatta cctgaccaag
     3241 tactccaaga aggataacgg accagtgatc aaaaagataa agtactatgg caacaaactt
     3301 aatgcgcatt tggacataac tgacgattac cccaattctc gaaacaaggt tgtgaagctc
     3361 tccctgaagc cttatagatt tgacgtgtac ctggataatg gggtttataa attcgtcacc
     3421 gtgaaaaatc tggacgtgat caaaaaggag aactattatg aagtaaactc aaagtgctat
     3481 gaggaggcga agaagctgaa gaagatctcc aatcaggccg agttcatcgc ttccttctat
     3541 aagaacgatc tcatcaagat caatggagag ctttatcgcg tcattggtgt gaacaatgac
     3601 ttgctgaaca ggatcgaagt caatatgata gacattacct accgggagta tctcgaaaac
     3661 atgaatgata aacggccgcc tcacatcatc aagacaatcg catctaaaac tcagtcaata
     3721 aaaaagtact ctaccgatat cctggggaat ctctatgaag tgaagtcaaa gaagcaccca
     3781 caaatcatta aaaaaggtgg atcccccaag aagaagagga aagtctcgag cgactacaaa
     3841 gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga tgacaagtct
     3901 ggtggttcta ctaatctgtc agatattatt gaaaaggaga ccggtaagca actggttatc
     3961 caggaatcca tcctcatgct cccagaggag gtggaagaag tcattgggaa caagccggaa
     4021 agcgatatac tcgtgcacac cgcctacgac gagagcaccg acgagaatgt catgcttctg
     4081 actagcgacg cccctgaata caagccttgg gctctggtca tacaggatag caacggtgag
     4141 aacaagatta agatgctctc tggtggttct cccaagaaga agaggaaagt ctaaccggtc
     4201 atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     4261 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     4321 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     4381 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     4441 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     4501 cgataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4561 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4621 cctagggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4681 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4741 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4801 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4861 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4921 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4981 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5041 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5101 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5161 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5221 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5281 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5341 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5401 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5461 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5521 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5581 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5641 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5701 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5761 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5821 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5881 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5941 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6001 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6061 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6121 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6181 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6241 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6301 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6361 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6421 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6481 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6541 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6601 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6661 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatcgatctc
     6721 ccgatcccct agggtcgact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     6781 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     6841 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     6901 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
     6961 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     7021 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     7081 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     7141 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatc
//
