LOCUS       sequence-438487-        7736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses monomeric mitochondrial cytosine base editor in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7736)
  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 7736)
  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: TC mDdCBE A1
FEATURES             Location/Qualifiers
     source          1..7736
                     /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             3520..3771
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      3968..4396
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4055..4074)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4265..4286
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4405..4425)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4410..4739
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4590..4725
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4652..4671
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4787..4834
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4853..5251
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5409..5530
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5446..5465)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5500..5519
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5579..5595)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5579..5595)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5592..5614)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5603..5619
                     /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(5627..5633,5634..5651,5652..5657))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5672..5693
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5810..5827)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5981..6569)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6061..6080)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6740..7531,7532..7600))
                     /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     7363..7382
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7601..7705)
                     /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 tggacatcgc ggatctgcgt
     1081 accctgggtt acagccagca gcagcaggag aagatcaagc caaaggtgcg cagcaccgtg
     1141 gcccagcacc atgaagctct ggtgggtcac ggcttcaccc acgcgcacat cgtggctctg
     1201 agccagcacc cagccgcgct gggtaccgtg gccgtgaaat atcaggacat gattgctgcc
     1261 ctgccagagg ctacccatga agcgattgtg ggtgtgggca agcagtggag cggtgctcgt
     1321 gcgctggagg ccctgctgac cgtggccggt gaactgcgtg gcccgccgct gcagctggat
     1381 accggccagc tgctgaaaat cgcgaaacgt ggcggtgtga ccgctgtgga agctgtgcat
     1441 gcctggcgta atgctctgac cggtgccccc ctgaacctga ccccggacca agtggtggct
     1501 atcgccagca acattggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     1561 ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag caacaatggc
     1621 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     1681 ctgactccgg accaagtggt ggctatcgcc agccacgatg gcggcaagca agcgctcgaa
     1741 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     1801 gtggctatcg ccagccacga tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     1861 ccggtgctgt gccaggacca tggcctgact ccggaccaag tggtggctat cgccagccac
     1921 gatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac
     1981 catggcctga ctccggacca agtggtggct atcgccagcc acgatggcgg caagcaagcg
     2041 ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac
     2101 caagtggtgg ctatcgccag caacaatggc ggcaagcaag cgctcgaaac ggtgcagcgg
     2161 ctgttgccgg tgctgtgcca ggaccatggc ctcacacccg atcaggtcgt ggctatcgcc
     2221 agcaacaatg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc
     2281 caggaccatg gcctgactcc ggaccaagtg gtggctatcg ccagccacga tggcggcaag
     2341 caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc
     2401 ccggaccaag tggtggctat cgccagcaac attggcggca agcaagcgct cgaaacggtg
     2461 cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct
     2521 atcgccagca acaatggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     2581 ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag caacaatggc
     2641 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     2701 ctgaccccgg accaagtggt ggctatcgcc agcaacggtg gcggcaagca agcgctcgaa
     2761 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     2821 gtggctatcg ccagcaacgg tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     2881 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     2941 ggtggcggca agcaagcgct cgaaagcatt gtggcccagc tgagccggcc tgatccggcg
     3001 ttggccgcgt tgaccaacga tcacctggtg gccctggcct gcctgggcgg tcgtccggcc
     3061 ctggatgcgg tgaagaaagg cctgggtgga tccggcagct acgccctggg tccgtatcag
     3121 attagcgccc cgcagctgcc agcatacaat ggtcagaccg tgggtacctt ctactatgtg
     3181 aacgacgctg gcggtctgga gggcaaggtg tttagcagcg gcggtccaac cccgtaccca
     3241 aactatgcca atgccggtca tgtggagagt cagagcgccc tgttcatgcg tgataacggc
     3301 atcagcgagg gtctggtgtt ccacaacaac ccggaaggca cctgcggttt ttgcgtgaac
     3361 atgaccgaga ccctgctgcc ggaaaacgcg aaaatgaccg tggtgccgcc ggaaggtgtc
     3421 attccagtga agcgcggcgc taccggtgaa accaaagtgt ttaccggtaa cagcaacggc
     3481 ccgaagagcc cgaccaaagg cggttgctct ggcggctcaa ctaatctgag cgacatcatt
     3541 gagaaggaga ctgggaaaca gctggtcatt caggagtcca tcctgatgct gcctgaggag
     3601 gtggaggaag tgatcggcaa caagccagag tctgacatcc tggtgcacac cgcctacgac
     3661 gagtccacag atgagaatgt gatgctgctg acctctgacg cccccgagta taagccttgg
     3721 gccctggtca tccaggattc taacggcgag aataagatca agatgctgtg ataaggggtc
     3781 tttgtcctct gtactgtctc tctccttgcc cctaacccaa aaagcttcat ttttctgtgt
     3841 aggctgcaca agagccttga ttgaagatat attctttctg aacagtattt aaggtttcca
     3901 ataaaatgta cacccctcag aacttctgag gcggaaagaa ccagctgggg ctctaggggg
     3961 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4021 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4081 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4141 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4201 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4261 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     4321 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     4381 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     4441 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     4501 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4561 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4621 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     4681 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     4741 agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc
     4801 atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa
     4861 gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat
     4921 ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt
     4981 cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg
     5041 cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa
     5101 caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg
     5161 gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt
     5221 gctgccctct ggttatgtgt gggagggcta agcacttcgt ggccgaggag caggactgac
     5281 acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5341 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5401 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5461 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5521 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     5581 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5641 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5701 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5761 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5821 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5881 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5941 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6001 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6061 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6121 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6181 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6241 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6301 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6361 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6421 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6481 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6541 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6601 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6661 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6721 agtaaacttg gtctgacagt taccaatgtt taatcagtga ggcacctatc tcagcgatct
     6781 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6841 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6901 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6961 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7021 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7081 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     7141 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     7201 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7261 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7321 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7381 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7441 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7501 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7561 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7621 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7681 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
