LOCUS       sequence-307668-        7143 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 7143)
  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 7143)
  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: G1397 DddAtox-C–SaKKH-Cas9(D10A)
FEATURES             Location/Qualifiers
     source          1..7143
                     /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             3763..3783
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3790..3855
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3868..4116
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4129..4149
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4158..4175
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4198..4215)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4204..4428
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4499..4515)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4499..4515)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4512..4534)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4523..4539
                     /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(4547..4553,4554..4571,4572..4577))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4592..4613
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4730..4747)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4901..5489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4981..5000)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5660..6451,6452..6520))
                     /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     6283..6302
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6521..6625)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6704..6723)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6895..7143,1..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 ggccattcca
      421 gtgaagcgcg gcgctaccgg tgaaaccaaa gtgtttaccg gtaacagcaa cagcccgaag
      481 agcccgacca aaggcggttg ctctggtggt tcttctggtg gttctagcgg cagcgagact
      541 cccgggacct cagagtccgc cacacccgaa agtagtggcg gcagcagcgg cggcagcggg
      601 aaacggaact acatcctggg gcttgccatt gggataacca gcgttggcta cggaattatt
      661 gattatgaga cacgcgatgt gattgacgcc ggggttaggc tgttcaaaga ggccaacgtt
      721 gaaaacaacg agggaagacg gagtaagcgc ggagcaagaa gactcaagcg cagacggaga
      781 catcggattc agagggtgaa aaagctgctc ttcgattaca atctcctgac cgatcatagt
      841 gagctgagcg gaatcaaccc ctacgaggcg cgagtgaaag ggctttccca gaagctgtcc
      901 gaagaggagt tctccgccgc gttgctgcac ctggccaaac ggaggggggt tcacaatgta
      961 aacgaagtgg aggaggacac gggcaatgaa cttagtacga aagaacagat cagtaggaac
     1021 tctaaggctc tcgaagagaa atacgtcgct gagttgcagc ttgagagact gaaaaaagac
     1081 ggcgaagtac gcggatctat taataggttc aagacttcag attacgtaaa ggaagccaag
     1141 cagctcctga aagtacagaa agcgtaccat cagctcgatc agagcttcat cgatacctac
     1201 atagatttgc tggagacacg gaggacatac tacgagggcc caggggaagg atctcctttt
     1261 gggtggaagg acatcaagga atggtacgag atgcttatgg gacattgtac atattttccg
     1321 gaggagctca ggagcgtcaa gtacgcctac aatgccgacc tgtacaatgc cctcaatgac
     1381 ctcaataacc tcgtgattac cagggacgag aacgagaagc tggagtacta tgaaaagttc
     1441 cagattatcg agaatgtgtt taagcagaag aagaagccga cacttaagca gattgcaaag
     1501 gaaatcctcg tgaatgagga agatatcaag ggatacagag tgacaagtac aggcaagccc
     1561 gagttcacaa atctgaaggt gtaccacgat attaaggaca taaccgcacg aaaggagata
     1621 atcgaaaacg ctgagctcct cgatcagatc gcaaaaattc ttaccatcta ccagtctagt
     1681 gaggacattc aggaggaact gactaatctg aacagtgagc tcacccaaga ggaaattgag
     1741 cagatttcaa acctgaaagg ctacaccggg acgcacaatc tgagcctcaa agcaatcaac
     1801 ctcattctgg atgaactttg gcacacaaat gacaaccaaa ttgccatatt caaccgcctg
     1861 aaactggtgc caaaaaaagt ggatctgtca cagcaaaagg aaatccctac aaccttggtt
     1921 gacgatttta ttctgtcccc cgttgtcaag cggagcttca tccagtcaat caaggtgatc
     1981 aatgccatca ttaaaaaata cggattgcca aacgatataa ttatcgagct tgcacgagag
     2041 aagaactcaa aggacgccca gaagatgatt aacgaaatgc agaagcgcaa ccgccagaca
     2101 aacgaacgca tagaggaaat tataagaaca accggcaaag agaatgccaa gtatctgatc
     2161 gagaaaatca agctgcacga catgcaagaa ggcaagtgcc tgtactctct ggaagctatc
     2221 ccactcgaag atctgctgaa taatccattc aattacgagg tggaccacat catccctaga
     2281 tccgtaagct ttgacaattc cttcaataac aaagttctgg ttaaacagga ggaaaattct
     2341 aaaaaaggga accggacccc gttccagtac ctgagctcca gtgacagcaa gattagctac
     2401 gagactttta agaaacatat tctgaatctg gccaaaggca aaggcaggat cagcaagacc
     2461 aagaaggagt acctcctcga agaacgcgac attaacagat ttagtgtgca gaaagatttc
     2521 atcaaccgaa accttgtcga tactcggtac gccacgagag gcctgatgaa tctcctcagg
     2581 agctacttcc gcgtcaataa tctggacgtt aaagtcaaga gcataaatgg gggattcacc
     2641 agctttctga ggagaaagtg gaagtttaag aaggaacgaa acaaaggata caagcaccat
     2701 gctgaggatg ctttgatcat cgctaacgcg gactttatct ttaaggaatg gaaaaagctg
     2761 gataaggcaa agaaagtgat ggaaaaccag atgttcgagg agaagcaggc agagtcaatg
     2821 cctgagatcg agacagagca ggaatacaag gaaattttca tcacccctca tcagattaaa
     2881 cacataaagg acttcaaaga ctataaatac tctcataggg tggacaaaaa acccaatcgc
     2941 aagctcatta atgacaccct gtactcaaca cggaaggatg ataaaggtaa taccttgatt
     3001 gtgaataatc ttaatggatt gtatgacaaa gataacgaca agctcaagaa gctgatcaac
     3061 aagtctccag agaagctcct tatgtatcac cacgacccac agacttatca gaaattgaaa
     3121 ctgatcatgg agcaatacgg ggatgagaag aacccactct acaaatatta tgaggaaaca
     3181 ggtaattacc tgaccaagta ctccaagaag gataacggac cagtgatcaa aaagataaag
     3241 tactatggca acaaacttaa tgcgcatttg gacataactg acgattaccc caattctcga
     3301 aacaaggttg tgaagctctc cctgaagcct tatagatttg acgtgtacct ggataatggg
     3361 gtttataaat tcgtcaccgt gaaaaatctg gacgtgatca aaaaggagaa ctattatgaa
     3421 gtaaactcaa agtgctatga ggaggcgaag aagctgaaga agatctccaa tcaggccgag
     3481 ttcatcgctt ccttctataa gaacgatctc atcaagatca atggagagct ttatcgcgtc
     3541 attggtgtga acaatgactt gctgaacagg atcgaagtca atatgataga cattacctac
     3601 cgggagtatc tcgaaaacat gaatgataaa cggccgcctc acatcatcaa gacaatcgca
     3661 tctaaaactc agtcaataaa aaagtactct accgatatcc tggggaatct ctatgaagtg
     3721 aagtcaaaga agcacccaca aatcattaaa aaaggtggat cccccaagaa gaagaggaaa
     3781 gtctcgagcg actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag
     3841 gatgacgatg acaagtctgg tggttctact aatctgtcag atattattga aaaggagacc
     3901 ggtaagcaac tggttatcca ggaatccatc ctcatgctcc cagaggaggt ggaagaagtc
     3961 attgggaaca agccggaaag cgatatactc gtgcacaccg cctacgacga gagcaccgac
     4021 gagaatgtca tgcttctgac tagcgacgcc cctgaataca agccttgggc tctggtcata
     4081 caggatagca acggtgagaa caagattaag atgctctctg gtggttctcc caagaagaag
     4141 aggaaagtct aaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct
     4201 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     4261 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     4321 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     4381 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     4441 aaagaaccag ctggggctcg ataccgtcga cctctagcta gagcttggcg taatcatggt
     4501 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4561 gaagcataaa gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4621 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4681 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4741 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4801 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4861 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4921 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4981 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5041 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5101 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5161 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5221 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5281 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5341 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5401 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5461 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5521 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5581 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5641 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5701 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5761 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5821 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5881 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5941 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6001 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6061 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6121 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6181 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6241 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6301 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6361 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6421 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6481 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6541 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6601 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     6661 gatcgggaga tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg
     6721 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     6781 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
     6841 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
     6901 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     6961 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     7021 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     7081 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     7141 atc
//
