LOCUS       sequence-308872-        7335 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 7335)
  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 7335)
  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-C–SaKKH-Cas9(D10A)
FEATURES             Location/Qualifiers
     source          1..7335
                     /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"
     CDS             3955..3975
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3982..4047
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4060..4308
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4321..4341
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4350..4367
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4390..4407)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4396..4620
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4691..4707)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4691..4707)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4704..4726)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4715..4731
                     /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(4739..4745,4746..4763,4764..4769))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4784..4805
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4922..4939)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5093..5681)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5173..5192)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5852..6643,6644..6712))
                     /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     6475..6494
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6713..6817)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6896..6915)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7087..7335,1..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 gccaaccccg
      421 tacccaaact atgccaatgc cggtcatgtg gagggtcaga gcgccctgtt catgcgtgat
      481 aacggcatca gcgagggtct ggtgttccac aacaacccgg aaggcacctg cggtttttgc
      541 gtgaacatga ccgagaccct gctgccggaa aacgcgaaaa tgaccgtggt gccgccggaa
      601 ggtgccattc cagtgaagcg cggcgctacc ggtgaaacca aagtgtttac cggtaacagc
      661 aacagcccga agagcccgac caaaggcggt tgctctggtg gttcttctgg tggttctagc
      721 ggcagcgaga ctcccgggac ctcagagtcc gccacacccg aaagtagtgg cggcagcagc
      781 ggcggcagcg ggaaacggaa ctacatcctg gggcttgcca ttgggataac cagcgttggc
      841 tacggaatta ttgattatga gacacgcgat gtgattgacg ccggggttag gctgttcaaa
      901 gaggccaacg ttgaaaacaa cgagggaaga cggagtaagc gcggagcaag aagactcaag
      961 cgcagacgga gacatcggat tcagagggtg aaaaagctgc tcttcgatta caatctcctg
     1021 accgatcata gtgagctgag cggaatcaac ccctacgagg cgcgagtgaa agggctttcc
     1081 cagaagctgt ccgaagagga gttctccgcc gcgttgctgc acctggccaa acggaggggg
     1141 gttcacaatg taaacgaagt ggaggaggac acgggcaatg aacttagtac gaaagaacag
     1201 atcagtagga actctaaggc tctcgaagag aaatacgtcg ctgagttgca gcttgagaga
     1261 ctgaaaaaag acggcgaagt acgcggatct attaataggt tcaagacttc agattacgta
     1321 aaggaagcca agcagctcct gaaagtacag aaagcgtacc atcagctcga tcagagcttc
     1381 atcgatacct acatagattt gctggagaca cggaggacat actacgaggg cccaggggaa
     1441 ggatctcctt ttgggtggaa ggacatcaag gaatggtacg agatgcttat gggacattgt
     1501 acatattttc cggaggagct caggagcgtc aagtacgcct acaatgccga cctgtacaat
     1561 gccctcaatg acctcaataa cctcgtgatt accagggacg agaacgagaa gctggagtac
     1621 tatgaaaagt tccagattat cgagaatgtg tttaagcaga agaagaagcc gacacttaag
     1681 cagattgcaa aggaaatcct cgtgaatgag gaagatatca agggatacag agtgacaagt
     1741 acaggcaagc ccgagttcac aaatctgaag gtgtaccacg atattaagga cataaccgca
     1801 cgaaaggaga taatcgaaaa cgctgagctc ctcgatcaga tcgcaaaaat tcttaccatc
     1861 taccagtcta gtgaggacat tcaggaggaa ctgactaatc tgaacagtga gctcacccaa
     1921 gaggaaattg agcagatttc aaacctgaaa ggctacaccg ggacgcacaa tctgagcctc
     1981 aaagcaatca acctcattct ggatgaactt tggcacacaa atgacaacca aattgccata
     2041 ttcaaccgcc tgaaactggt gccaaaaaaa gtggatctgt cacagcaaaa ggaaatccct
     2101 acaaccttgg ttgacgattt tattctgtcc cccgttgtca agcggagctt catccagtca
     2161 atcaaggtga tcaatgccat cattaaaaaa tacggattgc caaacgatat aattatcgag
     2221 cttgcacgag agaagaactc aaaggacgcc cagaagatga ttaacgaaat gcagaagcgc
     2281 aaccgccaga caaacgaacg catagaggaa attataagaa caaccggcaa agagaatgcc
     2341 aagtatctga tcgagaaaat caagctgcac gacatgcaag aaggcaagtg cctgtactct
     2401 ctggaagcta tcccactcga agatctgctg aataatccat tcaattacga ggtggaccac
     2461 atcatcccta gatccgtaag ctttgacaat tccttcaata acaaagttct ggttaaacag
     2521 gaggaaaatt ctaaaaaagg gaaccggacc ccgttccagt acctgagctc cagtgacagc
     2581 aagattagct acgagacttt taagaaacat attctgaatc tggccaaagg caaaggcagg
     2641 atcagcaaga ccaagaagga gtacctcctc gaagaacgcg acattaacag atttagtgtg
     2701 cagaaagatt tcatcaaccg aaaccttgtc gatactcggt acgccacgag aggcctgatg
     2761 aatctcctca ggagctactt ccgcgtcaat aatctggacg ttaaagtcaa gagcataaat
     2821 gggggattca ccagctttct gaggagaaag tggaagttta agaaggaacg aaacaaagga
     2881 tacaagcacc atgctgagga tgctttgatc atcgctaacg cggactttat ctttaaggaa
     2941 tggaaaaagc tggataaggc aaagaaagtg atggaaaacc agatgttcga ggagaagcag
     3001 gcagagtcaa tgcctgagat cgagacagag caggaataca aggaaatttt catcacccct
     3061 catcagatta aacacataaa ggacttcaaa gactataaat actctcatag ggtggacaaa
     3121 aaacccaatc gcaagctcat taatgacacc ctgtactcaa cacggaagga tgataaaggt
     3181 aataccttga ttgtgaataa tcttaatgga ttgtatgaca aagataacga caagctcaag
     3241 aagctgatca acaagtctcc agagaagctc cttatgtatc accacgaccc acagacttat
     3301 cagaaattga aactgatcat ggagcaatac ggggatgaga agaacccact ctacaaatat
     3361 tatgaggaaa caggtaatta cctgaccaag tactccaaga aggataacgg accagtgatc
     3421 aaaaagataa agtactatgg caacaaactt aatgcgcatt tggacataac tgacgattac
     3481 cccaattctc gaaacaaggt tgtgaagctc tccctgaagc cttatagatt tgacgtgtac
     3541 ctggataatg gggtttataa attcgtcacc gtgaaaaatc tggacgtgat caaaaaggag
     3601 aactattatg aagtaaactc aaagtgctat gaggaggcga agaagctgaa gaagatctcc
     3661 aatcaggccg agttcatcgc ttccttctat aagaacgatc tcatcaagat caatggagag
     3721 ctttatcgcg tcattggtgt gaacaatgac ttgctgaaca ggatcgaagt caatatgata
     3781 gacattacct accgggagta tctcgaaaac atgaatgata aacggccgcc tcacatcatc
     3841 aagacaatcg catctaaaac tcagtcaata aaaaagtact ctaccgatat cctggggaat
     3901 ctctatgaag tgaagtcaaa gaagcaccca caaatcatta aaaaaggtgg atcccccaag
     3961 aagaagagga aagtctcgag cgactacaaa gaccatgacg gtgattataa agatcatgac
     4021 atcgattaca aggatgacga tgacaagtct ggtggttcta ctaatctgtc agatattatt
     4081 gaaaaggaga ccggtaagca actggttatc caggaatcca tcctcatgct cccagaggag
     4141 gtggaagaag tcattgggaa caagccggaa agcgatatac tcgtgcacac cgcctacgac
     4201 gagagcaccg acgagaatgt catgcttctg actagcgacg cccctgaata caagccttgg
     4261 gctctggtca tacaggatag caacggtgag aacaagatta agatgctctc tggtggttct
     4321 cccaagaaga agaggaaagt ctaaccggtc atcatcacca tcaccattga gtttaaaccc
     4381 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     4441 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     4501 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     4561 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     4621 cttctgaggc ggaaagaacc agctggggct cgataccgtc gacctctagc tagagcttgg
     4681 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4741 acatacgagc cggaagcata aagtgtaaag cctagggtgc ctaatgagtg agctaactca
     4801 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4861 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4921 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4981 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5041 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5101 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5161 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5221 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5281 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5341 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5401 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5461 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5521 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5581 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5641 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5701 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5761 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5821 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5881 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5941 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6001 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6061 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6121 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6181 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6241 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6301 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6361 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6421 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6481 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6541 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6601 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6661 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6721 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6781 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6841 ctgacgtcga cggatcggga gatcgatctc ccgatcccct agggtcgact ctcagtacaa
     6901 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     6961 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     7021 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     7081 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     7141 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     7201 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     7261 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     7321 tacatcaagt gtatc
//
