LOCUS       sequence-438458-        7616 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 7616)
  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 7616)
  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: ND2 DdCBE C1
FEATURES             Location/Qualifiers
     source          1..7616
                     /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             3432..3683
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      3880..4308
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3967..3986)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4177..4198
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4317..4337)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4322..4651
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4502..4637
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4564..4583
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4699..4746
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4765..5163
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5321..5442
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5358..5377)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5412..5431
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5491..5507)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5491..5507)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5504..5526)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5515..5531
                     /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(5539..5545,5546..5563,5564..5569))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5584..5605
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5722..5739)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5893..6481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5973..5992)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6652..7443,7444..7512))
                     /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     7275..7294
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7513..7616,1))
                     /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 caacggtggc 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 ggtggcggca
     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 agcaacattg
     2161 gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg
     2221 gcctcacacc cgatcaggtc gtggctatcg ccagcaacgg 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 caacggtggc 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 tggcctgact ccggaccaag tggtggctat cgccagccac gatggcggca
     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 cgctcgaaac ggtgcagcgg ctgttgccgg
     3121 tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc agcaacaatg
     3181 gcggcaagca agcgctcgaa agcattgtgg cccagctgag ccggcctgat ccggcgttgg
     3241 ccgcgttgac caacgatcac ctggtggccc tggcctgcct gggcggtcgt ccggccctgg
     3301 atgcggtgaa gaaaggcctg ggtggatccg ccattccagt gaagcgcggc gctaccggtg
     3361 aaaccaaagt gtttaccggt aacagcaaca gcccgaagag cccgaccaaa ggcggttgct
     3421 ctggcggctc aactaatctg agcgacatca ttgagaagga gactgggaaa cagctggtca
     3481 ttcaggagtc catcctgatg ctgcctgagg aggtggagga agtgatcggc aacaagccag
     3541 agtctgacat cctggtgcac accgcctacg acgagtccac agatgagaat gtgatgctgc
     3601 tgacctctga cgcccccgag tataagcctt gggccctggt catccaggat tctaacggcg
     3661 agaataagat caagatgctg tgataagggg tctttgtcct ctgtactgtc tctctccttg
     3721 cccctaaccc aaaaagcttc atttttctgt gtaggctgca caagagcctt gattgaagat
     3781 atattctttc tgaacagtat ttaaggtttc caataaaatg tacacccctc agaacttctg
     3841 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3901 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3961 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4021 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4081 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4141 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4201 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     4261 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     4321 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4381 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     4441 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4501 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4561 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     4621 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     4681 cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     4741 tacgacaagg tgaggaacta aaccatggcc aagcctttgt ctcaagaaga atccaccctc
     4801 attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc
     4861 agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact
     4921 gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac
     4981 ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg
     5041 tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat
     5101 ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc
     5161 taagcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc
     5221 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5281 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5341 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5401 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5461 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5521 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5581 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5641 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5701 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5761 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5821 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5881 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5941 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6001 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6061 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6121 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6181 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6241 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6301 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6361 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6421 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6481 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6541 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6601 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6661 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6721 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6781 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6841 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6901 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6961 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7021 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7081 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7141 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7201 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7261 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7321 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7381 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7441 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7501 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7561 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgc
//
