LOCUS       sequence-391825-        7198 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Employ DdCBE-mediated mtDNA editing to ablate mtProtein(ND1) by 
            swapping Trp codon TGA with stop codon TAA in C6 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7198)
  AUTHORS   Tan L, Qi X, Kong W, Jin J, Lu D, Zhang X, Wang Y, Wang S, Dong W, 
            Shi X, Chen W, Wang J, Li K, Xie Y, Gao L, Guan F, Gao K, Li C, Wang
            C, Hu Z, Zhang L, Guo X, Shen B, Ma Y
  TITLE     A conditional knockout rat resource of mitochondrial protein-coding 
            genes via a DdCBE-induced premature stop codon.
  JOURNAL   Sci Adv. 2023 Apr 14;9(15):eadf2695. doi: 10.1126/sciadv.adf2695. 
            Epub 2023 Apr 14.
  PUBMED    37058569
REFERENCE   2  (bases 1 to 7198)
  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: "Sci Adv."; 
            date: "2023-04-14"; volume: "9(15):eadf2695. doi"; pages: " 
            10.1126/sciadv.adf2695. Epub 2023 Apr 14"
COMMENT     Sequence Label: pGL3-MTS-ND1-W86-L14-G1333C-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7198
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        388..591
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     542..562
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     636..655
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        636..654
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             863..886
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3110..3361
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     primer_bind     complement(3393..3410)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3399..3623
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3630..4140
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4059..4077
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4162..4761
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4162..4181)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4658..4678
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4908..5029
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4919..4938)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4973..4992
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5277..5294)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5448..6036)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5528..5547)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6207..6998,6999..7067))
                     /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     6830..6849
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7068..7172)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      421 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      481 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      541 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta
      601 actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta tagggagaca
      661 agctggctag accatgttgg ggtttgtggg tcgggtggcc gctgctccgg cctccggggc
      721 cttgcggaga ctcacccctt cagcgtcgct gcccccagct cagctcttac tgcgggccgc
      781 tccgacggcg gtccatcctg tcagggacta tgcggcgcaa acatctgaga gcggcggagg
      841 cgggagcccc ggcgcggccg ctgactacaa ggatgacgac gataaaggta gcgtggatct
      901 acgcacgctc ggctacagcc agcagcaaca ggagaagatc aaaccgaagg ttcgttcgac
      961 agtggcgcag caccacgagg cactggtcgg ccatgggttt acacacgcgc acatcgttgc
     1021 gctcagccaa cacccggcag cgttagggac cgtcgctgtc aagtatcagg acatgatcgc
     1081 agcgttgcca gaggcgacac acgaagcgat cgttggcgtc ggcaaacagt ggtccggcgc
     1141 acgcgctctg gaggccttgc tcacggtggc gggagagttg agaggtccac cgttacagtt
     1201 ggacacaggc caacttctca agattgcaaa acgtggcggc gtgaccgcag tggaggcagt
     1261 gcatgcatgg cgcaatgcac tgacgggtgc ccccctgaac ctgaccccgg agcaggtggt
     1321 ggccatcgct agtaatgggg gtggcaaaca ggctcttgag actgttcagc gccttctacc
     1381 agttctctgt caagcccacg gcctgacgcc cgagcaagtt gtagcgattg ctagtaatat
     1441 tggtggcaaa caggctcttg agacggttca gcgcctcctt ccagttctct gtcaagccca
     1501 cggactcacc ccagatcagg ttgtagcgat tgctagtcat gacggtggca aacaggctct
     1561 tgaaaccgtc caacgccttc taccagttct ctgtcaagcc cacggactaa ccccagcgca
     1621 agttgtagcg attgctagta atattggtgg caaacaggct cttgagacgg ttcagcgcct
     1681 ccttccagtt ctctgtcaag cccacggact caccccagat caagtggtag cgattgctag
     1741 tcatgacggt ggcaaacagg ctcttgaaac cgtccaacgc cttctaccag ttctctgtca
     1801 agcccacgga ctaaccccag cgcaagttgt agcgattgct agtaatgggg gtggcaaaca
     1861 ggctcttgag actgttcagc gccttctacc agttctctgt caagcccacg gcctgacgcc
     1921 cgagcaagtt gtagcgattg ctagtaatat tggtggcaaa caggctcttg agacggttca
     1981 gcgcctcctt ccagttctct gtcaagccca cggactcacc ccagatcaag ttgtagcgat
     2041 tgctagtaac aatggtggca aacaggctct tgaaaccgta cagcgcctac tgccagttct
     2101 ctgtcaagcc cacggtctga ctccggagca agttgtagca attgctagtc atgacggtgg
     2161 caaacaggct cttgaaaccg tccaacgcct tctaccagtt ctctgtcaag cccacggact
     2221 aaccccagcg caagttgtag cgattgctag taatgggggt ggcaaacagg ctcttgagac
     2281 tgttcagcgc cttctaccag ttctctgtca agcccacggc ctgacgcccg agcaagttgt
     2341 agcgattgct agtcatgacg gtggcaaaca ggctcttgaa accgtccaac gccttctacc
     2401 agttctctgt caagcccacg gactaacccc agcgcaagtt gtagcgattg ctagtaatgg
     2461 gggtggcaaa caggctcttg agactgttca gcgccttcta ccagttctct gtcaagccca
     2521 cggcctgacg cccgagcaag ttgtagcgat agccagtaat attggtggca aacaggctct
     2581 tgagacggtt cagcgcctcc ttccagttct ctgtcaagcc cacggactca ccccagatca
     2641 agttgtagcg attgcaagta atattggcgg caggccggcg ctggagagca ttgttgccca
     2701 gttatctcgc cctgatccgg cgttggccgc gttgaccaac gaccacctcg tcgccttggc
     2761 ctgcctcggc ggacgtcctg cgctggatgc agtgaaaaag ggattgggag gctccccaac
     2821 cccctaccct aactatgcca atgcaggaca cgtggaggga cagagcgccc tgttcatgcg
     2881 ggataacggc atctccgagg gcctggtgtt ccacaacaat cccgagggca cctgcggctt
     2941 ttgcgtgaac atgaccgaga cactgctgcc cgagaatgcc aagatgacag tggtgccacc
     3001 tgagggagca atccctgtga agagaggcgc caccggcgag acaaaggtgt ttaccggcaa
     3061 ctctaatagc cctaagtccc caacaaaggg cggctgctct ggaggcagca ccaacctgtc
     3121 tgacatcatc gagaaggaga caggcaagca gctggtcatc caggagagca tcctgatgct
     3181 gcccgaagaa gtcgaagaag tgatcggaaa caagcctgag agcgatatcc tggtccatac
     3241 cgcctacgac gagagtaccg acgaaaatgt gatgctgctg acatccgacg ccccagagta
     3301 taagccctgg gctctggtca tccaggattc caacggagag aacaaaatca aaatgctgta
     3361 atctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     3421 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3481 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3541 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3601 tggggatgcg gtgggctcta tggctcgagg gggttggggt tgcgcctttt ccaaggcagc
     3661 cctgggtttg cgcagggacg cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc
     3721 gaccctggga ctcgcacatt cttcacgtcc gttcgcagcg tcacccggat cttcgccgct
     3781 acccttgtgg gccccccggc gacgcttcct gctccgcccc taagtcggga aggttccttg
     3841 cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac gtctcactag taccctcgca
     3901 gacggacagc gccagggagc aatggcagcg cgccgaccgc gatgggctgt ggccaatagc
     3961 ggctgctcag cagggcgcgc cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg
     4021 tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct
     4081 ccggagcgca cgtcggcagt cggctccctc gttgaccgaa tcaccgacct ctctccccag
     4141 ggggatccac cggagcttac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     4201 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     4261 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     4321 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     4381 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     4441 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     4501 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     4561 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     4621 gccggggtgc ccgccttcct ggaaacctcc gcgccccgca acctcccctt ctacgagcgg
     4681 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     4741 acccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac cgaaaggagc
     4801 gcacgacccc atgcatcggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
     4861 agccactttc tagagtcggg gcggccggcc gcttcgagca gacatgataa gatacattga
     4921 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     4981 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     5041 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     5101 aaacctctac aaatgtggta aaatcgataa ggatccgtcg accgatgccc ttgagagcct
     5161 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     5221 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc cgcttcctcg
     5281 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5341 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5401 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5461 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5521 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5581 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5641 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5701 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5761 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5821 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5881 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5941 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6001 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6061 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6121 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6181 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6241 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6301 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggga cccacgctca
     6361 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6421 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6481 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6541 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6601 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6661 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6721 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6781 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6841 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6901 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6961 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7021 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7081 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7141 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctg
//
