LOCUS       sequence-391805-        7606 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 7606)
  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 7606)
  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-L18-G1333C-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7606
                     /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             3518..3769
                     /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(3801..3818)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3807..4031
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4038..4548
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4467..4485
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4570..5169
                     /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(4570..4589)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5066..5086
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5316..5437
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5327..5346)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5381..5400
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5685..5702)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5856..6444)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5936..5955)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6615..7406,7407..7475))
                     /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     7238..7257
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7476..7580)
                     /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 agtaacaatg gtggcaaaca ggctcttgaa accgtacagc gcctactgcc
     1381 agttctctgt caagcccacg gtctgactcc ggagcaagtt 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 atgggggtgg caaacaggct cttgagactg ttcagcgcct
     1681 tctaccagtt ctctgtcaag cccacggcct gacgcccgag 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 tgctagtaat attggtggca aacaggctct tgagacggtt cagcgcctcc ttccagttct
     2101 ctgtcaagcc cacggactca ccccagatca agttgtagca attgctagta acaatggtgg
     2161 caaacaggct cttgaaaccg tacagcgcct actgccagtt ctctgtcaag cccacggtct
     2221 gactccggag caagttgtag cgattgctag tcatgacggt ggcaaacagg ctcttgaaac
     2281 cgtccaacgc cttctaccag ttctctgtca agcccacgga ctaaccccag cgcaagttgt
     2341 agcgattgct agtcatgacg gtggcaaaca ggctcttgaa accgtccaac gccttctacc
     2401 agttctctgt caagcccacg gactaacccc agcgcaagtt 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 taatattggt ggcaaacagg ctcttgagac ggttcagcgc ctccttccag ttctctgtca
     2821 agcccacgga ctcaccccag atcaagttgt agcgattgct agtaatattg gtggcaaaca
     2881 ggctcttgag acggttcagc gcctccttcc agttctctgt caagcccacg gactcacccc
     2941 agatcaagtt gtagcgattg cttccaatat tggtggcaaa caggctcttg agacggttca
     3001 gcgcctcctt ccagttctct gtcaagccca cggactcacc ccagatcaag ttgtagcgat
     3061 tgcctctaat attggcggca ggccggcgct ggagagcatt gttgcccagt tatctcgccc
     3121 tgatccggcg ttggccgcgt tgaccaacga ccacctcgtc gccttggcct gcctcggcgg
     3181 acgtcctgcg ctggatgcag tgaaaaaggg attgggaggc tccccaaccc cctaccctaa
     3241 ctatgccaat gcaggacacg tggagggaca gagcgccctg ttcatgcggg ataacggcat
     3301 ctccgagggc ctggtgttcc acaacaatcc cgagggcacc tgcggctttt gcgtgaacat
     3361 gaccgagaca ctgctgcccg agaatgccaa gatgacagtg gtgccacctg agggagcaat
     3421 ccctgtgaag agaggcgcca ccggcgagac aaaggtgttt accggcaact ctaatagccc
     3481 taagtcccca acaaagggcg gctgctctgg aggcagcacc aacctgtctg acatcatcga
     3541 gaaggagaca ggcaagcagc tggtcatcca ggagagcatc ctgatgctgc ccgaagaagt
     3601 cgaagaagtg atcggaaaca agcctgagag cgatatcctg gtccataccg cctacgacga
     3661 gagtaccgac gaaaatgtga tgctgctgac atccgacgcc ccagagtata agccctgggc
     3721 tctggtcatc caggattcca acggagagaa caaaatcaaa atgctgtaat ctagagggcc
     3781 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3841 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3901 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3961 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     4021 gggctctatg gctcgagggg gttggggttg cgccttttcc aaggcagccc tgggtttgcg
     4081 cagggacgcg gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggact
     4141 cgcacattct tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc
     4201 cccccggcga cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg
     4261 tgccggacgt gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc
     4321 cagggagcaa tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca
     4381 gggcgcgccg agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta
     4441 gtgtgggccc tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg
     4501 tcggcagtcg gctccctcgt tgaccgaatc accgacctct ctccccaggg ggatccaccg
     4561 gagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     4621 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     4681 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     4741 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     4801 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     4861 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     4921 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     4981 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     5041 gccttcctgg aaacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     5101 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     5161 ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc acgaccccat
     5221 gcatcggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccactttcta
     5281 gagtcggggc ggccggccgc ttcgagcaga catgataaga tacattgatg agtttggaca
     5341 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     5401 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     5461 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     5521 atgtggtaaa atcgataagg atccgtcgac cgatgccctt gagagccttc aacccagtca
     5581 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     5641 tcatgcaact cgtaggacag gtgccggcag cgctcttccg cttcctcgct cactgactcg
     5701 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5761 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5821 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5881 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5941 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6001 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     6061 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6121 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6181 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6241 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6301 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6361 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6421 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6481 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6541 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6601 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6661 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6721 ttaccatctg gccccagtgc tgcaatgata ccgcgggacc cacgctcacc ggctccagat
     6781 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6841 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6901 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6961 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7021 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     7081 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7141 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7201 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7261 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7321 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7381 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7441 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7501 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7561 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctg
//
