LOCUS       sequence-438696-        7748 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 7748)
  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 7748)
  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 alphaDdCBE T2 (DddA6-N)
FEATURES             Location/Qualifiers
     source          1..7748
                     /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             3564..3815
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      4012..4440
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4099..4118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4309..4330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4449..4469)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4454..4783
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4634..4769
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4696..4715
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4831..4878
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4897..5295
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5453..5574
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5490..5509)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5544..5563
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5623..5639)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5623..5639)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5636..5658)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5647..5663
                     /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(5671..5677,5678..5695,5696..5701))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5716..5737
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5854..5871)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6025..6613)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6105..6124)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6784..7575,7576..7644))
                     /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     7407..7426
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7645..7748,1))
                     /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 gcctgacccc ggaccaagtg gtggctatcg
     1741 ccagcaacaa tggcggcaag caagcgctcg aaacggtgca gcggctgttg ccggtgctgt
     1801 gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac ggtggcggca
     1861 agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac catggcctga
     1921 ccccggacca agtggtggct atcgccagca acggtggcgg caagcaagcg ctcgaaacgg
     1981 tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac caagtggtgg
     2041 ctatcgccag caacggtggc ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg
     2101 tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc agcaacggtg
     2161 gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg
     2221 gcctcacacc cgatcaggtc gtggctatcg ccagcaacat tggcggcaag caagcgctcg
     2281 aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc ccggaccaag
     2341 tggtggctat cgccagcaac ggtggcggca agcaagcgct cgaaacggtg cagcggctgt
     2401 tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct atcgccagca
     2461 acaatggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg
     2521 accatggcct gactccggac caagtggtgg ctatcgccag ccacgatggc ggcaagcaag
     2581 cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg
     2641 accaagtggt ggctatcgcc agcaacaatg gcggcaagca agcgctcgaa acggtgcagc
     2701 ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg gtggctatcg
     2761 ccagcaacat tggcggcaag caagcgctcg aaacggtgca gcggctgttg ccggtgctgt
     2821 gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac ggtggcggca
     2881 agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac catggcctga
     2941 ccccggacca agtggtggct atcgccagca acggtggcgg caagcaagcg ctcgaaacgg
     3001 tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac caagtggtgg
     3061 ctatcgccag caacattggc ggcaagcaag cgctcgaaag cattgtggcc cagctgagcc
     3121 ggcctgatcc ggcgttggcc gcgttgacca acgatcacct ggtggccctg gcctgcctgg
     3181 gcggtcgtcc ggccctggat gcggtgaaga aaggcctggg tggatccggc agctacgccc
     3241 tgggtccgta tcagattagc gccccgcagc tgccagctta caatggtcgg accgtgggta
     3301 ccttctacta tgtgaacgac gcgggcggtc tggagagcaa ggtgtttatc agcggcggtc
     3361 caaccccgta cccaaactat gccaatgccg gtcatgtgga gggtcagagc gccctgttca
     3421 tgcgtgataa cggcatcagc gagggtctgg tgttccacaa caacccggaa ggcacctgcg
     3481 gtttttgcgt gaacatgatc gagaccctgc tgccggaaaa cgcgaaaatg accgtggtgc
     3541 cgccggaagg ttctggcggc tcaactaatc tgagcgacat cattgagaag gagactggga
     3601 aacagctggt cattcaggag tccatcctga tgctgcctga ggaggtggag gaagtgatcg
     3661 gcaacaagcc agagtctgac atcctggtgc acaccgccta cgacgagtcc acagatgaga
     3721 atgtgatgct gctgacctct gacgcccccg agtataagcc ttgggccctg gtcatccagg
     3781 attctaacgg cgagaataag atcaagatgc tgtgataagg ggtctttgtc ctctgtactg
     3841 tctctctcct tgcccctaac ccaaaaagct tcatttttct gtgtaggctg cacaagagcc
     3901 ttgattgaag atatattctt tctgaacagt atttaaggtt tccaataaaa tgtacacccc
     3961 tcagaacttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     4021 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     4081 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4141 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4201 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     4261 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4321 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     4381 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4441 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     4501 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     4561 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     4621 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4681 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     4741 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     4801 tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc
     4861 ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagccttt gtctcaagaa
     4921 gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac
     4981 tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta
     5041 tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg
     5101 gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc
     5161 ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg
     5221 aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat
     5281 gtgtgggagg gctaagcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt
     5341 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5401 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     5461 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5521 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5581 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5641 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5701 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5761 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5821 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5881 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5941 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6001 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6061 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6121 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6181 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6241 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6301 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6361 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6421 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6481 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6541 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6601 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6661 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6721 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6781 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6841 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6901 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6961 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7021 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7081 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7141 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7201 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7261 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7321 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7381 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7441 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7501 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7561 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7621 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7681 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7741 ggttccgc
//
