LOCUS       sequence-307635-        7377 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 7377)
  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 7377)
  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-N–SaKKH-Cas9(D10A)
FEATURES             Location/Qualifiers
     source          1..7377
                     /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             3997..4017
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4024..4089
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4102..4350
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4363..4383
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4392..4409
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4432..4449)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4438..4662
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4733..4749)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4733..4749)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4746..4768)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4757..4773
                     /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(4781..4787,4788..4805,4806..4811))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4826..4847
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4964..4981)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5135..5723)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5215..5234)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5894..6685,6686..6754))
                     /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     6517..6536
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6755..6859)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6938..6957)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7129..7377,1..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 ggtccaaccc cgtacccaaa ctatgccaat gccggtcatg tggagggtca gagcgccctg
      601 ttcatgcgtg ataacggcat cagcgagggt ctggtgttcc acaacaaccc ggaaggcacc
      661 tgcggttttt gcgtgaacat gaccgagacc ctgctgccgg aaaacgcgaa aatgaccgtg
      721 gtgccgccgg aaggttctgg tggttcttct ggtggttcta gcggcagcga gactcccggg
      781 acctcagagt ccgccacacc cgaaagtagt ggcggcagca gcggcggcag cgggaaacgg
      841 aactacatcc tggggcttgc cattgggata accagcgttg gctacggaat tattgattat
      901 gagacacgcg atgtgattga cgccggggtt aggctgttca aagaggccaa cgttgaaaac
      961 aacgagggaa gacggagtaa gcgcggagca agaagactca agcgcagacg gagacatcgg
     1021 attcagaggg tgaaaaagct gctcttcgat tacaatctcc tgaccgatca tagtgagctg
     1081 agcggaatca acccctacga ggcgcgagtg aaagggcttt cccagaagct gtccgaagag
     1141 gagttctccg ccgcgttgct gcacctggcc aaacggaggg gggttcacaa tgtaaacgaa
     1201 gtggaggagg acacgggcaa tgaacttagt acgaaagaac agatcagtag gaactctaag
     1261 gctctcgaag agaaatacgt cgctgagttg cagcttgaga gactgaaaaa agacggcgaa
     1321 gtacgcggat ctattaatag gttcaagact tcagattacg taaaggaagc caagcagctc
     1381 ctgaaagtac agaaagcgta ccatcagctc gatcagagct tcatcgatac ctacatagat
     1441 ttgctggaga cacggaggac atactacgag ggcccagggg aaggatctcc ttttgggtgg
     1501 aaggacatca aggaatggta cgagatgctt atgggacatt gtacatattt tccggaggag
     1561 ctcaggagcg tcaagtacgc ctacaatgcc gacctgtaca atgccctcaa tgacctcaat
     1621 aacctcgtga ttaccaggga cgagaacgag aagctggagt actatgaaaa gttccagatt
     1681 atcgagaatg tgtttaagca gaagaagaag ccgacactta agcagattgc aaaggaaatc
     1741 ctcgtgaatg aggaagatat caagggatac agagtgacaa gtacaggcaa gcccgagttc
     1801 acaaatctga aggtgtacca cgatattaag gacataaccg cacgaaagga gataatcgaa
     1861 aacgctgagc tcctcgatca gatcgcaaaa attcttacca tctaccagtc tagtgaggac
     1921 attcaggagg aactgactaa tctgaacagt gagctcaccc aagaggaaat tgagcagatt
     1981 tcaaacctga aaggctacac cgggacgcac aatctgagcc tcaaagcaat caacctcatt
     2041 ctggatgaac tttggcacac aaatgacaac caaattgcca tattcaaccg cctgaaactg
     2101 gtgccaaaaa aagtggatct gtcacagcaa aaggaaatcc ctacaacctt ggttgacgat
     2161 tttattctgt cccccgttgt caagcggagc ttcatccagt caatcaaggt gatcaatgcc
     2221 atcattaaaa aatacggatt gccaaacgat ataattatcg agcttgcacg agagaagaac
     2281 tcaaaggacg cccagaagat gattaacgaa atgcagaagc gcaaccgcca gacaaacgaa
     2341 cgcatagagg aaattataag aacaaccggc aaagagaatg ccaagtatct gatcgagaaa
     2401 atcaagctgc acgacatgca agaaggcaag tgcctgtact ctctggaagc tatcccactc
     2461 gaagatctgc tgaataatcc attcaattac gaggtggacc acatcatccc tagatccgta
     2521 agctttgaca attccttcaa taacaaagtt ctggttaaac aggaggaaaa ttctaaaaaa
     2581 gggaaccgga ccccgttcca gtacctgagc tccagtgaca gcaagattag ctacgagact
     2641 tttaagaaac atattctgaa tctggccaaa ggcaaaggca ggatcagcaa gaccaagaag
     2701 gagtacctcc tcgaagaacg cgacattaac agatttagtg tgcagaaaga tttcatcaac
     2761 cgaaaccttg tcgatactcg gtacgccacg agaggcctga tgaatctcct caggagctac
     2821 ttccgcgtca ataatctgga cgttaaagtc aagagcataa atgggggatt caccagcttt
     2881 ctgaggagaa agtggaagtt taagaaggaa cgaaacaaag gatacaagca ccatgctgag
     2941 gatgctttga tcatcgctaa cgcggacttt atctttaagg aatggaaaaa gctggataag
     3001 gcaaagaaag tgatggaaaa ccagatgttc gaggagaagc aggcagagtc aatgcctgag
     3061 atcgagacag agcaggaata caaggaaatt ttcatcaccc ctcatcagat taaacacata
     3121 aaggacttca aagactataa atactctcat agggtggaca aaaaacccaa tcgcaagctc
     3181 attaatgaca ccctgtactc aacacggaag gatgataaag gtaatacctt gattgtgaat
     3241 aatcttaatg gattgtatga caaagataac gacaagctca agaagctgat caacaagtct
     3301 ccagagaagc tccttatgta tcaccacgac ccacagactt atcagaaatt gaaactgatc
     3361 atggagcaat acggggatga gaagaaccca ctctacaaat attatgagga aacaggtaat
     3421 tacctgacca agtactccaa gaaggataac ggaccagtga tcaaaaagat aaagtactat
     3481 ggcaacaaac ttaatgcgca tttggacata actgacgatt accccaattc tcgaaacaag
     3541 gttgtgaagc tctccctgaa gccttataga tttgacgtgt acctggataa tggggtttat
     3601 aaattcgtca ccgtgaaaaa tctggacgtg atcaaaaagg agaactatta tgaagtaaac
     3661 tcaaagtgct atgaggaggc gaagaagctg aagaagatct ccaatcaggc cgagttcatc
     3721 gcttccttct ataagaacga tctcatcaag atcaatggag agctttatcg cgtcattggt
     3781 gtgaacaatg acttgctgaa caggatcgaa gtcaatatga tagacattac ctaccgggag
     3841 tatctcgaaa acatgaatga taaacggccg cctcacatca tcaagacaat cgcatctaaa
     3901 actcagtcaa taaaaaagta ctctaccgat atcctgggga atctctatga agtgaagtca
     3961 aagaagcacc cacaaatcat taaaaaaggt ggatccccca agaagaagag gaaagtctcg
     4021 agcgactaca aagaccatga cggtgattat aaagatcatg acatcgatta caaggatgac
     4081 gatgacaagt ctggtggttc tactaatctg tcagatatta ttgaaaagga gaccggtaag
     4141 caactggtta tccaggaatc catcctcatg ctcccagagg aggtggaaga agtcattggg
     4201 aacaagccgg aaagcgatat actcgtgcac accgcctacg acgagagcac cgacgagaat
     4261 gtcatgcttc tgactagcga cgcccctgaa tacaagcctt gggctctggt catacaggat
     4321 agcaacggtg agaacaagat taagatgctc tctggtggtt ctcccaagaa gaagaggaaa
     4381 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg
     4441 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     4501 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     4561 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     4621 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     4681 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4741 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4801 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4861 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4921 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4981 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5041 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5101 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5161 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5221 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5281 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5341 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5401 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5461 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5521 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5581 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5641 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5701 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5761 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5821 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5881 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5941 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6001 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6061 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6121 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6181 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6241 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6301 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6361 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6421 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6481 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6541 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6601 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6661 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6721 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6781 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6841 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     6901 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat
     6961 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     7021 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     7081 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     7141 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     7201 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     7261 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     7321 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc
//
