LOCUS       sequence-391823-        7354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Employ DdCBE-mediated mtDNA editing to ablate mtProtein(ND4) 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 7354)
  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 7354)
  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-ND4-W24-R17-G1333N-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7354
                     /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             3266..3517
                     /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(3549..3566)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3555..3779
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3786..4296
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4215..4233
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4318..4917
                     /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(4318..4337)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4814..4834
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5064..5185
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5075..5094)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5129..5148
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5433..5450)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5604..6192)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5684..5703)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6363..7154,7155..7223))
                     /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     6986..7005
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7224..7328)
                     /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 ctagtaacaa
     1441 tggtggcaaa caggctcttg aaaccgtaca gcgcctactg ccagttctct gtcaagccca
     1501 cggtctgact ccggagcagg ttgtagcgat tgctagtaat attggtggca aacaggctct
     1561 tgagacggtt cagcgcctcc ttccagttct ctgtcaagcc cacggactca ccccagatca
     1621 agttgtagcg attgctagta atattggtgg caaacaggct cttgagacgg ttcagcgcct
     1681 ccttccagtt ctctgtcaag cccacggact caccccagat caagtggtag cgattgctag
     1741 taatattggt ggcaaacagg ctcttgagac ggttcagcgc ctccttccag ttctctgtca
     1801 agcccacgga ctcaccccag atcaagttgt agcgattgct agtaacaatg gtggcaaaca
     1861 ggctcttgaa accgtacagc gcctactgcc agttctctgt caagcccacg gtctgactcc
     1921 ggagcaagtt gtagcgattg ctagtcatga cggtggcaaa caggctcttg aaaccgtcca
     1981 acgccttcta ccagttctct gtcaagccca cggactaacc ccagcgcaag ttgtagcgat
     2041 tgctagtaat gggggtggca aacaggctct tgagactgtt cagcgccttc taccagttct
     2101 ctgtcaagcc cacggcctga cgcccgagca 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 ctagtaacaa
     2461 tggtggcaaa caggctcttg aaaccgtaca gcgcctactg ccagttctct gtcaagccca
     2521 cggtctgact ccggagcaag ttgtagcgat agccagtaac aatggtggca aacaggctct
     2581 tgaaaccgta cagcgcctac tgccagttct ctgtcaagcc cacggtctga ctccggagca
     2641 agttgtagcg attgctagta atattggtgg caaacaggct cttgagacgg ttcagcgcct
     2701 ccttccagtt ctctgtcaag cccacggact caccccagat caagttgtag cgattgcaag
     2761 taacaatggt ggcaaacagg ctcttgaaac cgtacagcgc ctactgccag ttctctgtca
     2821 agcccacggt ctgactccgg agcaagttgt agcgattgct agtaacaatg gtggcaaaca
     2881 ggctcttgaa accgtacagc gcctactgcc agttctctgt caagcccacg gtctgactcc
     2941 ggagcaagtt gtagcgattg cttccaatgg gggcggcagg ccggcgctgg agagcattgt
     3001 tgcccagtta tctcgccctg atccggcgtt ggccgcgttg accaacgacc acctcgtcgc
     3061 cttggcctgc ctcggcggac gtcctgcgct ggatgcagtg aaaaagggat tgggaggctc
     3121 cggctcctac gccctgggcc catatcagat ctctgcccca cagctgccag catacaacgg
     3181 acagaccgtg ggcacattct actatgtgaa tgacgccggc ggcctggagt ctaaggtgtt
     3241 tagctccggc ggctctggag gcagcaccaa cctgtctgac atcatcgaga aggagacagg
     3301 caagcagctg gtcatccagg agagcatcct gatgctgccc gaagaagtcg aagaagtgat
     3361 cggaaacaag cctgagagcg atatcctggt ccataccgcc tacgacgaga gtaccgacga
     3421 aaatgtgatg ctgctgacat ccgacgcccc agagtataag ccctgggctc tggtcatcca
     3481 ggattccaac ggagagaaca aaatcaaaat gctgtaatct agagggcccg tttaaacccg
     3541 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3601 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3661 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3721 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3781 tcgagggggt tggggttgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc
     3841 tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggactcg cacattcttc
     3901 acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg
     3961 cttcctgctc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga
     4021 caaacggaag ccgcacgtct cactagtacc ctcgcagacg gacagcgcca gggagcaatg
     4081 gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcagg gcgcgccgag
     4141 agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg
     4201 ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc
     4261 tccctcgttg accgaatcac cgacctctct ccccaggggg atccaccgga gcttaccatg
     4321 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     4381 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     4441 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     4501 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     4561 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     4621 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     4681 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     4741 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggaa
     4801 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     4861 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc
     4921 ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgc atcggtacct
     4981 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttctaga gtcggggcgg
     5041 ccggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     5101 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     5161 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     5221 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat
     5281 cgataaggat ccgtcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     5341 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     5401 taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5461 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5521 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5581 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5641 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5701 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5761 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5821 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5881 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5941 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6001 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6061 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6121 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6181 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6241 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6301 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6361 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6421 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6481 cccagtgctg caatgatacc gcgggaccca cgctcaccgg ctccagattt atcagcaata
     6541 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6601 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6661 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6721 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6781 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6841 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6901 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6961 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7021 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7081 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7141 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7201 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7261 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7321 gttccgcgca catttccccg aaaagtgcca cctg
//
