LOCUS       sequence-438452-        5387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mitochondrially targeted TALE-free DddAtox 1397C-UGI in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5387)
  AUTHORS   Castillo SR, Simone BW, Clark KJ, Devaux P, Ekker SC
  TITLE     Unconstrained Precision Mitochondrial Genome Editing with 
            alphaDdCBEs.
  JOURNAL   Hum Gene Ther. 2024 Sep 24. doi: 10.1089/hum.2024.073.
  PUBMED    39212664
REFERENCE   2  (bases 1 to 5387)
  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: "Hum Gene 
            Ther. 2024 Sep 24. doi: 10.1089/hum.2024.073."
COMMENT     Sequence Label: TALE-free DddAtox 1397C
FEATURES             Location/Qualifiers
     source          1..5387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(76..95)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        267..646
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        647..850
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     801..821
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     895..914
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        895..913
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             936..1013
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MASVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1026..1091
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1203..1454
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      1651..2079
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1738..1757)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1948..1969
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2088..2108)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2093..2422
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2273..2408
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2335..2354
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2470..2517
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2536..2934
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3092..3213
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3129..3148)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3183..3202
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3262..3278)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3262..3278)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3275..3297)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3286..3302
                     /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(3310..3316,3317..3334,3335..3340))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3355..3376
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3493..3510)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3664..4252)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3744..3763)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4423..5214,5215..5283))
                     /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     5046..5065
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5284..5387,1))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
       61 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
      121 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
      181 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
      241 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
      301 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      361 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      421 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      481 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      541 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      601 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      661 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      721 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      781 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      841 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
      901 gactcactat agggagaccc aagctggcta gcaccatggc ctccgttctg accccgctgc
      961 tgctgcgtgg cctgaccggc tccgctcgtc gtctgccagt tccgcgtgcg aaaatccatt
     1021 ccctggacta caaagaccat gacggtgatt ataaagatca tgacatcgat tacaaggatg
     1081 acgatgacaa gatgggatcc gccattccag tgaagcgcgg cgctaccggt gaaaccaaag
     1141 tgtttaccgg taacagcaac agcccgaaga gcccgaccaa aggcggttgc tctggcggct
     1201 caactaatct gagcgacatc attgagaagg agactgggaa acagctggtc attcaggagt
     1261 ccatcctgat gctgcctgag gaggtggagg aagtgatcgg caacaagcca gagtctgaca
     1321 tcctggtgca caccgcctac gacgagtcca cagatgagaa tgtgatgctg ctgacctctg
     1381 acgcccccga gtataagcct tgggccctgg tcatccagga ttctaacggc gagaataaga
     1441 tcaagatgct gtgataaggg gtctttgtcc tctgtactgt ctctctcctt gcccctaacc
     1501 caaaaagctt catttttctg tgtaggctgc acaagagcct tgattgaaga tatattcttt
     1561 ctgaacagta tttaaggttt ccaataaaat gtacacccct cagaacttct gaggcggaaa
     1621 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     1681 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     1741 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     1801 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     1861 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1921 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1981 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2041 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     2101 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2161 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2221 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     2281 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     2341 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     2401 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     2461 tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag
     2521 gtgaggaact aaaccatggc caagcctttg tctcaagaag aatccaccct cattgaaaga
     2581 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct
     2641 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct
     2701 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt
     2761 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag
     2821 gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg
     2881 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaagcactt
     2941 cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg ccgccttcta
     3001 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3061 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     3121 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3181 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     3241 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3301 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3361 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3421 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3481 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3541 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3601 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3661 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3721 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3781 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3841 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3901 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgtg ccttatccgg
     3961 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4021 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4081 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4141 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4201 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4261 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4321 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4381 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4441 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4501 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4561 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4621 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4681 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4741 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4801 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4861 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4921 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4981 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5041 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5101 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5161 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5221 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5281 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5341 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgc
//
