LOCUS       sequence-391799-        7402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Employ DdCBE-mediated mtDNA editing to ablate mtProtein(ND2) 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 7402)
  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 7402)
  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-ND2-W30-L16-G1333C-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7402
                     /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             3314..3565
                     /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(3597..3614)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3603..3827
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3834..4344
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4263..4281
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4366..4965
                     /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(4366..4385)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4862..4882
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5112..5233
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5123..5142)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5177..5196
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5481..5498)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5652..6240)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5732..5751)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6411..7202,7203..7271))
                     /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     7034..7053
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7272..7376)
                     /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 agtcatgacg gtggcaaaca ggctcttgaa accgtccaac gccttctacc
     1381 agttctctgt caagcccacg gactaacccc agcgcaagtt gtagcgattg ctagtaatgg
     1441 gggtggcaaa caggctcttg agactgttca gcgccttcta ccagttctct gtcaagccca
     1501 cggcctgacg cccgagcagg ttgtagcgat tgctagtaat attggtggca aacaggctct
     1561 tgagacggtt cagcgcctcc ttccagttct ctgtcaagcc cacggactca ccccagatca
     1621 agttgtagcg attgctagta acaatggtgg caaacaggct cttgaaaccg tacagcgcct
     1681 actgccagtt ctctgtcaag cccacggtct gactccggag 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 ctagtcatga cggtggcaaa caggctcttg aaaccgtcca
     1981 acgccttcta ccagttctct gtcaagccca cggactaacc ccagcgcaag ttgtagcgat
     2041 tgctagtcat gacggtggca aacaggctct tgaaaccgtc caacgccttc taccagttct
     2101 ctgtcaagcc cacggactaa ccccagcgca agttgtagca attgctagta atattggtgg
     2161 caaacaggct cttgagacgg ttcagcgcct ccttccagtt ctctgtcaag cccacggact
     2221 caccccagat caagttgtag cgattgctag taatattggt ggcaaacagg ctcttgagac
     2281 ggttcagcgc ctccttccag ttctctgtca agcccacgga ctcaccccag atcaagttgt
     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 gggggtggca aacaggctct
     2581 tgagactgtt cagcgccttc taccagttct ctgtcaagcc cacggcctga cgcccgagca
     2641 agttgtagcg attgctagta atattggtgg caaacaggct cttgagacgg ttcagcgcct
     2701 ccttccagtt ctctgtcaag cccacggact caccccagat caagttgtag cgattgcaag
     2761 tcatgacggt ggcaaacagg ctcttgaaac cgtccaacgc cttctaccag ttctctgtca
     2821 agcccacgga ctaaccccag cgcaagttgt agcgattgct tccaatgggg gcggcaggcc
     2881 ggcgctggag agcattgttg cccagttatc tcgccctgat ccggcgttgg ccgcgttgac
     2941 caacgaccac ctcgtcgcct tggcctgcct cggcggacgt cctgcgctgg atgcagtgaa
     3001 aaagggattg ggaggctccc caacccccta ccctaactat gccaatgcag gacacgtgga
     3061 gggacagagc gccctgttca tgcgggataa cggcatctcc gagggcctgg tgttccacaa
     3121 caatcccgag ggcacctgcg gcttttgcgt gaacatgacc gagacactgc tgcccgagaa
     3181 tgccaagatg acagtggtgc cacctgaggg agcaatccct gtgaagagag gcgccaccgg
     3241 cgagacaaag gtgtttaccg gcaactctaa tagccctaag tccccaacaa agggcggctg
     3301 ctctggaggc agcaccaacc tgtctgacat catcgagaag gagacaggca agcagctggt
     3361 catccaggag agcatcctga tgctgcccga agaagtcgaa gaagtgatcg gaaacaagcc
     3421 tgagagcgat atcctggtcc ataccgccta cgacgagagt accgacgaaa atgtgatgct
     3481 gctgacatcc gacgccccag agtataagcc ctgggctctg gtcatccagg attccaacgg
     3541 agagaacaaa atcaaaatgc tgtaatctag agggcccgtt taaacccgct gatcagcctc
     3601 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3661 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3721 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3781 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctc gagggggttg
     3841 gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt
     3901 ggttccggga aacgcagcgg cgccgaccct gggactcgca cattcttcac gtccgttcgc
     3961 agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg
     4021 cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc
     4081 gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga
     4141 ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg
     4201 gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg
     4261 cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac
     4321 cgaatcaccg acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag
     4381 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     4441 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     4501 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     4561 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     4621 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     4681 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     4741 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     4801 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggaaac ctccgcgccc
     4861 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     4921 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacgccc gccccacgac
     4981 ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat cggtaccttt aagaccaatg
     5041 acttacaagg cagctgtaga tcttagccac tttctagagt cggggcggcc ggccgcttcg
     5101 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     5161 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     5221 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     5281 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc
     5341 gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     5401 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     5461 cggcagcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5521 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5581 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5641 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5701 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5761 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5821 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5881 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5941 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6001 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6061 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6121 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6181 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6241 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6301 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6361 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6421 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6481 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6541 atgataccgc gggacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6601 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6661 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6721 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6781 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6841 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6901 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6961 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7021 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7081 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7141 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7201 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7261 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7321 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7381 tttccccgaa aagtgccacc tg
//
