LOCUS       sequence-438463-        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: CO1 alphaDdCBE C1
FEATURES             Location/Qualifiers
     source          1..7514
                     /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             3298..3549
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      3746..4174
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3833..3852)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4043..4064
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4183..4203)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4188..4517
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4368..4503
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4430..4449
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4565..4612
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4631..5029
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5187..5308
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5224..5243)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5278..5297
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5357..5373)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5357..5373)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5370..5392)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5381..5397
                     /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(5405..5411,5412..5429,5430..5435))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5450..5471
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5588..5605)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5759..6347)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5839..5858)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6518..7309,7310..7378))
                     /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     7141..7160
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7379..7483)
                     /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 agagaggagc cggtgctcgt
     1321 gcgctggagg ccctgctgac cgtggccggt gaactgcgtg gcccgccgct gcagctggat
     1381 accggccagc tgctgaaaat cgcgaaacgt ggcggtgtga ccgctgtgga agctgtgcat
     1441 gcctggcgta atgctctgac cggtgccccc ctgaacctga ccccggacca agtggtggct
     1501 atcgccagca acggtggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     1561 ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag caacggtggc
     1621 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     1681 ctgactccgg accaagtggt ggctatcgcc agccacgatg gcggcaagca agcgctcgaa
     1741 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg
     1801 gtggctatcg ccagcaacgg tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     1861 ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac
     1921 ggtggcggca 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 agcaacattg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc
     2281 caggaccatg gcctgactcc ggaccaagtg gtggctatcg ccagccacga tggcggcaag
     2341 caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc
     2401 ccggaccaag tggtggctat cgccagccac gatggcggca agcaagcgct cgaaacggtg
     2461 cagcggctgt tgccggtgct gtgccaggac catggcctga ctccggacca agtggtggct
     2521 atcgccagcc acgatggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg
     2581 ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag ccacgatggc
     2641 ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc
     2701 ctgaccccgg accaagtggt ggctatcgcc agcaacaatg gcggcaagca agcgctcgaa
     2761 acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgactcc ggaccaagtg
     2821 gtggctatcg ccagccacga tggcggcaag caagcgctcg aaacggtgca gcggctgttg
     2881 ccggtgctgt gccaggacca tggcctgact ccggaccaag tggtggctat cgccagccac
     2941 gatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac
     3001 catggcctga ccccggacca agtggtggct atcgccagca acaatggcgg caagcaagcg
     3061 ctcgaaagca ttgtggccca gctgagccgg cctgatccgg cgttggccgc gttgaccaac
     3121 gatcacctgg tggccctggc ctgcctgggc ggtcgtccgg ccctggatgc ggtgaagaaa
     3181 ggcctgggtg gatccgccat tccagtgaag cgcggcgcta ccggtgaaac caaagtgttt
     3241 accggtaaca gcaacagccc gaagagcccg accaaaggcg gttgctctgg cggctcaact
     3301 aatctgagcg acatcattga gaaggagact gggaaacagc tggtcattca ggagtccatc
     3361 ctgatgctgc ctgaggaggt ggaggaagtg atcggcaaca agccagagtc tgacatcctg
     3421 gtgcacaccg cctacgacga gtccacagat gagaatgtga tgctgctgac ctctgacgcc
     3481 cccgagtata agccttgggc cctggtcatc caggattcta acggcgagaa taagatcaag
     3541 atgctgtgat aaggggtctt tgtcctctgt actgtctctc tccttgcccc taacccaaaa
     3601 agcttcattt ttctgtgtag gctgcacaag agccttgatt gaagatatat tctttctgaa
     3661 cagtatttaa ggtttccaat aaaatgtaca cccctcagaa cttctgaggc ggaaagaacc
     3721 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3781 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     3841 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3901 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3961 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4021 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4081 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4141 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     4201 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     4261 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     4321 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     4381 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     4441 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     4501 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc
     4561 acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
     4621 gaactaaacc atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac
     4681 ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc
     4741 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc
     4801 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt
     4861 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct
     4921 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc
     4981 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag cacttcgtgg
     5041 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa
     5101 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5161 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     5221 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5281 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     5341 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5401 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5461 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5521 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5581 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5641 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5701 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5761 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5821 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5881 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5941 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6001 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6061 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6121 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6181 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6241 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6301 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6361 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6421 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6481 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6541 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6601 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6661 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6721 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6781 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6841 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6901 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6961 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7021 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7081 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7141 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7201 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7261 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7321 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7381 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7441 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7501 tgccacctga cgtc
//
