LOCUS       sequence-438491-        7412 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 7412)
  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 7412)
  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 DdCBE A1
FEATURES             Location/Qualifiers
     source          1..7412
                     /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             3228..3479
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      3676..4104
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3763..3782)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3973..3994
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4113..4133)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4118..4447
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4298..4433
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4360..4379
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4495..4542
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4561..4959
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5117..5238
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5154..5173)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5208..5227
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5287..5303)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5287..5303)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5300..5322)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5311..5327
                     /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(5335..5341,5342..5359,5360..5365))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5380..5401
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5518..5535)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5689..6277)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5769..5788)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6448..7239,7240..7308))
                     /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     7071..7090
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7309..7412,1))
                     /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 caacattggc ggcaagcaag
     1561 cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg
     1621 accaagtggt ggctatcgcc agcaacaatg gcggcaagca agcgctcgaa acggtgcagc
     1681 ggctgttgcc ggtgctgtgc caggaccatg gcctgactcc ggaccaagtg gtggctatcg
     1741 ccagccacga tggcggcaag caagcgctcg aaacggtgca gcggctgttg ccggtgctgt
     1801 gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagccac gatggcggca
     1861 agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac catggcctga
     1921 ctccggacca agtggtggct atcgccagcc acgatggcgg caagcaagcg ctcgaaacgg
     1981 tgcagcggct gttgccggtg ctgtgccagg accatggcct gactccggac caagtggtgg
     2041 ctatcgccag ccacgatggc ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg
     2101 tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc agcaacaatg
     2161 gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg
     2221 gcctcacacc cgatcaggtc gtggctatcg ccagcaacaa tggcggcaag caagcgctcg
     2281 aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgact ccggaccaag
     2341 tggtggctat cgccagccac gatggcggca 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 agcaacaatg gcggcaagca agcgctcgaa acggtgcagc
     2701 ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg gtggctatcg
     2761 ccagcaacgg 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 ctcgaaagca
     3001 ttgtggccca gctgagccgg cctgatccgg cgttggccgc gttgaccaac gatcacctgg
     3061 tggccctggc ctgcctgggc ggtcgtccgg ccctggatgc ggtgaagaaa ggcctgggtg
     3121 gatccgccat tccagtgaag cgcggcgcta ccggtgaaac caaagtgttt accggtaaca
     3181 gcaacagccc gaagagcccg accaaaggcg gttgctctgg cggctcaact aatctgagcg
     3241 acatcattga gaaggagact gggaaacagc tggtcattca ggagtccatc ctgatgctgc
     3301 ctgaggaggt ggaggaagtg atcggcaaca agccagagtc tgacatcctg gtgcacaccg
     3361 cctacgacga gtccacagat gagaatgtga tgctgctgac ctctgacgcc cccgagtata
     3421 agccttgggc cctggtcatc caggattcta acggcgagaa taagatcaag atgctgtgat
     3481 aaggggtctt tgtcctctgt actgtctctc tccttgcccc taacccaaaa agcttcattt
     3541 ttctgtgtag gctgcacaag agccttgatt gaagatatat tctttctgaa cagtatttaa
     3601 ggtttccaat aaaatgtaca cccctcagaa cttctgaggc ggaaagaacc agctggggct
     3661 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3721 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3781 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3841 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3901 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3961 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4021 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4081 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     4141 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4201 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4261 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4321 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4381 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4441 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac
     4501 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc
     4561 atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc
     4621 aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc
     4681 cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg
     4741 gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga
     4801 aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg
     4861 catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt
     4921 cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag cacttcgtgg ccgaggagca
     4981 ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     5041 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     5101 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5161 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5221 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     5281 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5341 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5401 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5461 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5521 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5581 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5641 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5701 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5761 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5821 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5881 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5941 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6001 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6061 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6121 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6181 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6241 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6301 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6361 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6421 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6481 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6541 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6601 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6661 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6721 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6781 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6841 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6901 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6961 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7021 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7081 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7141 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7201 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7261 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7321 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7381 tatttagaaa aataaacaaa taggggttcc gc
//
