LOCUS       sequence-438699-        7514 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mitochondrial cytosine base editor arm in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7514)
  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 7514)
  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: TL1 alphaL_DdCBE 1 (left, DddA11-C)
FEATURES             Location/Qualifiers
     source          1..7514
                     /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             3330..3581
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      3778..4206
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3865..3884)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4075..4096
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4215..4235)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4220..4549
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4400..4535
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4462..4481
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4597..4644
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4663..5061
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5219..5340
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5256..5275)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5310..5329
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5389..5405)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5389..5405)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5402..5424)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5413..5429
                     /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(5437..5443,5444..5461,5462..5467))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5482..5503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5620..5637)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5791..6379)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5871..5890)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6550..7341,7342..7410))
                     /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     7173..7192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7411..7514,1))
                     /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 gatggacatc gcggatctgc gtaccctggg ttacagccag cagcagcagg
     1141 agaagatcaa gccaaaggtg cgcagcaccg tggcccagca ccatgaagct ctggtgggtc
     1201 acggcttcac ccacgcgcac atcgtggctc tgagccagca cccagccgcg ctgggtaccg
     1261 tggccgtgaa atatcaggac atgattgctg ccctgccaga ggctacccat gaagcgattg
     1321 tgggtgtggg caagagagga gccggtgctc gtgcgctgga ggccctgctg accgtggccg
     1381 gtgaactgcg tggcccgccg ctgcagctgg ataccggcca gctgctgaaa atcgcgaaac
     1441 gtggcggtgt gaccgctgtg gaagctgtgc atgcctggcg taatgctctg accggtgccc
     1501 ccctgaacct gaccccggac caagtggtgg ctatcgccag caacattggc ggcaagcaag
     1561 cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg
     1621 accaagtggt ggctatcgcc agcaacattg gcggcaagca agcgctcgaa acggtgcagc
     1681 ggctgttgcc ggtgctgtgc caggaccatg gcctgactcc ggaccaagtg gtggctatcg
     1741 ccagccacga tggcggcaag caagcgctcg aaacggtgca gcggctgttg ccggtgctgt
     1801 gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac attggcggca
     1861 agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac catggcctga
     1921 ccccggacca agtggtggct atcgccagca acaatggcgg caagcaagcg ctcgaaacgg
     1981 tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac caagtggtgg
     2041 ctatcgccag caacaatggc ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg
     2101 tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc agcaacaatg
     2161 gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg
     2221 gcctcacacc cgatcaggtc gtggctatcg ccagcaacgg tggcggcaag caagcgctcg
     2281 aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc ccggaccaag
     2341 tggtggctat cgccagcaac ggtggcggca agcaagcgct cgaaacggtg cagcggctgt
     2401 tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct atcgccagca
     2461 acggtggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg
     2521 accatggcct gaccccggac caagtggtgg ctatcgccag caacaatggc ggcaagcaag
     2581 cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg
     2641 accaagtggt ggctatcgcc agcaacggtg gcggcaagca agcgctcgaa acggtgcagc
     2701 ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg gtggctatcg
     2761 ccagcaacgg tggcggcaag caagcgctcg aaacggtgca gcggctgttg ccggtgctgt
     2821 gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac attggcggca
     2881 agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac catggcctga
     2941 ccccggacca agtggtggct atcgccagca acattggcgg caagcaagcg ctcgaaacgg
     3001 tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac caagtggtgg
     3061 ctatcgccag caacaatggc ggcaagcaag cgctcgaaag cattgtggcc cagctgagcc
     3121 ggcctgatcc ggcgttggcc gcgttgacca acgatcacct ggtggccctg gcctgcctgg
     3181 gcggtcgtcc ggccctggat gcggtgaaga aaggcctggg tggatccgcc attccagtga
     3241 agcgcggcgc taccggtgaa accaaagtgt ttatcggtaa cagcaacagc ccgaagagcc
     3301 cgaccaaagg cggttgctct ggcggctcaa ctaatctgag cgacatcatt gagaaggaga
     3361 ctgggaaaca gctggtcatt caggagtcca tcctgatgct gcctgaggag gtggaggaag
     3421 tgatcggcaa caagccagag tctgacatcc tggtgcacac cgcctacgac gagtccacag
     3481 atgagaatgt gatgctgctg acctctgacg cccccgagta taagccttgg gccctggtca
     3541 tccaggattc taacggcgag aataagatca agatgctgtg ataaggggtc tttgtcctct
     3601 gtactgtctc tctccttgcc cctaacccaa aaagcttcat ttttctgtgt aggctgcaca
     3661 agagccttga ttgaagatat attctttctg aacagtattt aaggtttcca ataaaatgta
     3721 cacccctcag aacttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     3781 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3841 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3901 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3961 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     4021 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4081 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     4141 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4201 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     4261 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     4321 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     4381 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4441 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     4501 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     4561 agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag
     4621 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gcctttgtct
     4681 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
     4741 gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
     4801 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
     4861 gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc
     4921 ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc
     4981 atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct
     5041 ggttatgtgt gggagggcta agcacttcgt ggccgaggag caggactgac acgtgctacg
     5101 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     5161 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     5221 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5281 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5341 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5401 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5461 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5521 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5581 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5641 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5701 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5761 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5821 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5881 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5941 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6001 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6061 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6121 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6181 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6241 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6301 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6361 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6421 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6481 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6541 gtctgacagt taccaatgtt taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6601 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6661 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6721 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6781 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6841 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6901 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6961 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7021 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7081 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7141 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7201 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7261 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7321 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7381 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7441 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7501 aataggggtt ccgc
//
