LOCUS       sequence-391821-        7504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Employ DdCBE-mediated mtDNA editing to ablate mtProtein(COX3) 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 7504)
  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 7504)
  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-COX3-W16-L17-G1333C-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7504
                     /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             3416..3667
                     /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(3699..3716)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3705..3929
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3936..4446
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4365..4383
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4468..5067
                     /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(4468..4487)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4964..4984
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5214..5335
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5225..5244)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5279..5298
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5583..5600)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5754..6342)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5834..5853)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6513..7304,7305..7373))
                     /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     7136..7155
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7374..7478)
                     /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 agtaatattg gtggcaaaca ggctcttgag acggttcagc gcctccttcc
     1381 agttctctgt caagcccacg gactcacccc agatcaagtt gtagcgattg ctagtcatga
     1441 cggtggcaaa caggctcttg aaaccgtcca acgccttcta ccagttctct gtcaagccca
     1501 cggactaacc ccagcgcagg 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 taatgggggt ggcaaacagg ctcttgagac tgttcagcgc cttctaccag ttctctgtca
     1801 agcccacggc ctgacgcccg agcaagttgt agcgattgct agtaatattg gtggcaaaca
     1861 ggctcttgag acggttcagc gcctccttcc agttctctgt caagcccacg gactcacccc
     1921 agatcaagtt gtagcgattg ctagtaatgg gggtggcaaa caggctcttg agactgttca
     1981 gcgccttcta ccagttctct gtcaagccca cggcctgacg cccgagcaag ttgtagcgat
     2041 tgctagtaat attggtggca aacaggctct tgagacggtt cagcgcctcc ttccagttct
     2101 ctgtcaagcc cacggactca ccccagatca agttgtagca attgctagta acaatggtgg
     2161 caaacaggct cttgaaaccg tacagcgcct actgccagtt ctctgtcaag cccacggtct
     2221 gactccggag caagttgtag cgattgctag taatgggggt ggcaaacagg ctcttgagac
     2281 tgttcagcgc cttctaccag ttctctgtca agcccacggc ctgacgcccg agcaagttgt
     2341 agcgattgct agtaatattg gtggcaaaca ggctcttgag acggttcagc gcctccttcc
     2401 agttctctgt caagcccacg gactcacccc agatcaagtt gtagcgattg ctagtaatat
     2461 tggtggcaaa caggctcttg agacggttca gcgcctcctt ccagttctct gtcaagccca
     2521 cggactcacc ccagatcaag ttgtagcgat agccagtaat attggtggca aacaggctct
     2581 tgagacggtt cagcgcctcc ttccagttct ctgtcaagcc cacggactca ccccagatca
     2641 agttgtagcg attgctagtc atgacggtgg caaacaggct cttgaaaccg tccaacgcct
     2701 tctaccagtt ctctgtcaag cccacggact aaccccagcg caagttgtag cgattgcaag
     2761 tcatgacggt ggcaaacagg ctcttgaaac cgtccaacgc cttctaccag ttctctgtca
     2821 agcccacgga ctaaccccag cgcaagttgt agcgattgct agtcatgacg gtggcaaaca
     2881 ggctcttgaa accgtccaac gccttctacc agttctctgt caagcccacg gactaacccc
     2941 agcgcaagtt gtagcgattg cttccaatat tggcggcagg ccggcgctgg agagcattgt
     3001 tgcccagtta tctcgccctg atccggcgtt ggccgcgttg accaacgacc acctcgtcgc
     3061 cttggcctgc ctcggcggac gtcctgcgct ggatgcagtg aaaaagggat tgggaggctc
     3121 cccaaccccc taccctaact atgccaatgc aggacacgtg gagggacaga gcgccctgtt
     3181 catgcgggat aacggcatct ccgagggcct ggtgttccac aacaatcccg agggcacctg
     3241 cggcttttgc gtgaacatga ccgagacact gctgcccgag aatgccaaga tgacagtggt
     3301 gccacctgag ggagcaatcc ctgtgaagag aggcgccacc ggcgagacaa aggtgtttac
     3361 cggcaactct aatagcccta agtccccaac aaagggcggc tgctctggag gcagcaccaa
     3421 cctgtctgac atcatcgaga aggagacagg caagcagctg gtcatccagg agagcatcct
     3481 gatgctgccc gaagaagtcg aagaagtgat cggaaacaag cctgagagcg atatcctggt
     3541 ccataccgcc tacgacgaga gtaccgacga aaatgtgatg ctgctgacat ccgacgcccc
     3601 agagtataag ccctgggctc tggtcatcca ggattccaac ggagagaaca aaatcaaaat
     3661 gctgtaatct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     3721 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3781 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3841 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3901 gcatgctggg gatgcggtgg gctctatggc tcgagggggt tggggttgcg ccttttccaa
     3961 ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc
     4021 ggcgccgacc ctgggactcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc
     4081 gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt
     4141 tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc
     4201 ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc
     4261 aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag
     4321 gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc
     4381 aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct
     4441 ccccaggggg atccaccgga gcttaccatg accgagtaca agcccacggt gcgcctcgcc
     4501 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     4561 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     4621 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     4681 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     4741 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     4801 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     4861 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     4921 gagcgcgccg gggtgcccgc cttcctggaa acctccgcgc cccgcaacct ccccttctac
     4981 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     5041 tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa
     5101 aggagcgcac gaccccatgc atcggtacct ttaagaccaa tgacttacaa ggcagctgta
     5161 gatcttagcc actttctaga gtcggggcgg ccggccgctt cgagcagaca tgataagata
     5221 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     5281 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     5341 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag
     5401 caagtaaaac ctctacaaat gtggtaaaat cgataaggat ccgtcgaccg atgcccttga
     5461 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     5521 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctcttccgct
     5581 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5641 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5701 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5761 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5821 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5881 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5941 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6001 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6061 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6121 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6181 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6241 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6301 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6361 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6421 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6481 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6541 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6601 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgggaccca
     6661 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6721 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6781 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6841 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6901 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6961 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7021 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7081 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7141 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7201 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7261 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7321 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7381 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7441 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7501 cctg
//
