LOCUS       sequence-438635-        5621 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mitochondrially targeted TALE-free DddAtox 1397N-UGI in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5621)
  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 5621)
  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 1397N
FEATURES             Location/Qualifiers
     source          1..5621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             904..981
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MASVLTPLLLRGLTGSARRLPVPRAK"
     CDS             994..1059
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1405..1656
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      1853..2281
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1940..1959)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2150..2171
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2290..2310)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2295..2624
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2475..2610
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2537..2556
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2672..2719
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2738..3136
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3294..3415
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3331..3350)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3385..3404
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3464..3480)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3464..3480)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3477..3499)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3488..3504
                     /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(3512..3518,3519..3536,3537..3542))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3557..3578
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3695..3712)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3866..4454)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3946..3965)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4625..5416,5417..5485))
                     /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     5248..5267
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5486..5590)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 accatggcct ccgttctgac cccgctgctg ctgcgtggcc tgaccggctc cgctcgtcgt
      961 ctgccagttc cgcgtgcgaa aatccattcc ctggactaca aagaccatga cggtgattat
     1021 aaagatcatg acatcgatta caaggatgac gatgacaaga tgggatccgg cagctacgcc
     1081 ctgggtccgt atcagattag cgccccgcag ctgccagctt acaatggtca gaccgtgggt
     1141 accttctact atgtgaacga cgcgggcggt ctggagagca aggtgtttag cagcggcggt
     1201 ccaaccccgt acccaaacta tgccaatgcc ggtcatgtgg agggtcagag cgccctgttc
     1261 atgcgtgata acggcatcag cgagggtctg gtgttccaca acaacccgga aggcacctgc
     1321 ggtttttgcg tgaacatgac cgagaccctg ctgccggaaa acgcgaaaat gaccgtggtg
     1381 ccgccggaag gttctggcgg ctcaactaat ctgagcgaca tcattgagaa ggagactggg
     1441 aaacagctgg tcattcagga gtccatcctg atgctgcctg aggaggtgga ggaagtgatc
     1501 ggcaacaagc cagagtctga catcctggtg cacaccgcct acgacgagtc cacagatgag
     1561 aatgtgatgc tgctgacctc tgacgccccc gagtataagc cttgggccct ggtcatccag
     1621 gattctaacg gcgagaataa gatcaagatg ctgtgataag gggtctttgt cctctgtact
     1681 gtctctctcc ttgcccctaa cccaaaaagc ttcatttttc tgtgtaggct gcacaagagc
     1741 cttgattgaa gatatattct ttctgaacag tatttaaggt ttccaataaa atgtacaccc
     1801 ctcagaactt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     1861 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1921 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1981 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2041 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2101 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2161 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2221 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2281 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     2341 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     2401 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     2461 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     2521 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     2581 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     2641 gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat
     2701 cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagcctt tgtctcaaga
     2761 agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga
     2821 ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt
     2881 atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc
     2941 ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag
     3001 cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt
     3061 gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta
     3121 tgtgtgggag ggctaagcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt
     3181 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     3241 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     3301 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3361 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     3421 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     3481 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     3541 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3601 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3661 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3721 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3781 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3841 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3901 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3961 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4021 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4081 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4141 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4201 tatcgccact tgcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4261 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4321 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4381 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4441 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4501 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4561 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4621 acagttacca atgtttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4681 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4741 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4801 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4861 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4921 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4981 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5041 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5101 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5161 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5221 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5281 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5341 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5401 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5461 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5521 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5581 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
