LOCUS       sequence-438718-        7646 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 7646)
  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 7646)
  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: ND4 DdCBE T2
FEATURES             Location/Qualifiers
     source          1..7646
                     /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             3462..3713
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      3910..4338
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3997..4016)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4207..4228
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4347..4367)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4352..4681
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4532..4667
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4594..4613
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4729..4776
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4795..5193
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5351..5472
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5388..5407)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5442..5461
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5521..5537)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5521..5537)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5534..5556)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5545..5561
                     /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(5569..5575,5576..5593,5594..5599))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5614..5635
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5752..5769)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5923..6511)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6003..6022)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6682..7473,7474..7542))
                     /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     7305..7324
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7543..7646,1))
                     /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 caagcagtgg agcggtgctc gtgcgctgga ggccctgctg accgtggccg
     1381 gtgaactgcg tggcccgccg ctgcagctgg ataccggcca gctgctgaaa atcgcgaaac
     1441 gtggcggtgt gaccgctgtg gaagctgtgc atgcctggcg taatgctctg accggtgccc
     1501 ccctgaacct gaccccggac caagtggtgg ctatcgccag caacaatggc ggcaagcaag
     1561 cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg
     1621 accaagtggt ggctatcgcc agcaacggtg gcggcaagca agcgctcgaa acggtgcagc
     1681 ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg gtggctatcg
     1741 ccagcaacat 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 caacggtggc ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg
     2101 tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc agcaacattg
     2161 gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg
     2221 gcctcacacc cgatcaggtc gtggctatcg ccagcaacaa tggcggcaag caagcgctcg
     2281 aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc ccggaccaag
     2341 tggtggctat cgccagcaac aatggcggca agcaagcgct cgaaacggtg cagcggctgt
     2401 tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct atcgccagca
     2461 acattggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg
     2521 accatggcct gaccccggac caagtggtgg ctatcgccag caacaatggc ggcaagcaag
     2581 cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg
     2641 accaagtggt ggctatcgcc agcaacattg gcggcaagca agcgctcgaa acggtgcagc
     2701 ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg gtggctatcg
     2761 ccagcaacaa tggcggcaag caagcgctcg aaacggtgca gcggctgttg ccggtgctgt
     2821 gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac ggtggcggca
     2881 agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac catggcctga
     2941 ccccggacca agtggtggct atcgccagca acaatggcgg caagcaagcg ctcgaaagca
     3001 ttgtggccca gctgagccgg cctgatccgg cgttggccgc gttgaccaac gatcacctgg
     3061 tggccctggc ctgcctgggc ggtcgtccgg ccctggatgc ggtgaagaaa ggcctgggtg
     3121 gatccggcag ctacgccctg ggtccgtatc agattagcgc cccgcagctg ccagcttaca
     3181 atggtcagac cgtgggtacc ttctactatg tgaacgacgc gggcggtctg gagagcaagg
     3241 tgtttagcag cggcggtcca accccgtacc caaactatgc caatgccggt catgtggagg
     3301 gtcagagcgc cctgttcatg cgtgataacg gcatcagcga gggtctggtg ttccacaaca
     3361 acccggaagg cacctgcggt ttttgcgtga acatgaccga gaccctgctg ccggaaaacg
     3421 cgaaaatgac cgtggtgccg ccggaaggtt ctggcggctc aactaatctg agcgacatca
     3481 ttgagaagga gactgggaaa cagctggtca ttcaggagtc catcctgatg ctgcctgagg
     3541 aggtggagga agtgatcggc aacaagccag agtctgacat cctggtgcac accgcctacg
     3601 acgagtccac agatgagaat gtgatgctgc tgacctctga cgcccccgag tataagcctt
     3661 gggccctggt catccaggat tctaacggcg agaataagat caagatgctg tgataagggg
     3721 tctttgtcct ctgtactgtc tctctccttg cccctaaccc aaaaagcttc atttttctgt
     3781 gtaggctgca caagagcctt gattgaagat atattctttc tgaacagtat ttaaggtttc
     3841 caataaaatg tacacccctc agaacttctg aggcggaaag aaccagctgg ggctctaggg
     3901 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3961 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4021 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4081 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     4141 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4201 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     4261 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     4321 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     4381 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     4441 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4501 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4561 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4621 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     4681 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa
     4741 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc
     4801 aagcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc
     4861 atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc
     4921 ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg
     4981 ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag
     5041 aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct
     5101 gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa
     5161 ttgctgccct ctggttatgt gtgggagggc taagcacttc gtggccgagg agcaggactg
     5221 acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     5281 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     5341 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5401 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5461 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     5521 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5581 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5641 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5701 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5761 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5821 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5881 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5941 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6001 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6061 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6121 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6181 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6241 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6301 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6361 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6421 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6481 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6541 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6601 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6661 tgagtaaact tggtctgaca gttaccaatg tttaatcagt gaggcaccta tctcagcgat
     6721 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6781 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6841 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6901 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6961 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7021 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7081 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7141 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7201 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7261 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7321 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7381 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7441 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7501 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7561 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7621 gaaaaataaa caaatagggg ttccgc
//
