LOCUS       sequence-391822-        7300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Employ DdCBE-mediated mtDNA editing to ablate mtProtein(ND5) 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 7300)
  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 7300)
  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-ND5-W68-L15-G1333C-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7300
                     /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             3212..3463
                     /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(3495..3512)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3501..3725
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3732..4242
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4161..4179
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4264..4863
                     /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(4264..4283)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4760..4780
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5010..5131
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5021..5040)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5075..5094
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5379..5396)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5550..6138)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5630..5649)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6309..7100,7101..7169))
                     /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     6932..6951
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7170..7274)
                     /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 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 atgggggtgg caaacaggct cttgagactg ttcagcgcct
     1681 tctaccagtt ctctgtcaag cccacggcct gacgcccgag caagtggtag cgattgctag
     1741 taatattggt ggcaaacagg ctcttgagac ggttcagcgc ctccttccag ttctctgtca
     1801 agcccacgga ctcaccccag atcaagttgt 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 gggggtggca aacaggctct tgagactgtt cagcgccttc taccagttct
     2101 ctgtcaagcc cacggcctga cgcccgagca agttgtagca attgctagta atattggtgg
     2161 caaacaggct cttgagacgg ttcagcgcct ccttccagtt ctctgtcaag cccacggact
     2221 caccccagat caagttgtag cgattgctag tcatgacggt ggcaaacagg ctcttgaaac
     2281 cgtccaacgc cttctaccag ttctctgtca agcccacgga ctaaccccag cgcaagttgt
     2341 agcgattgct agtaatgggg gtggcaaaca ggctcttgag actgttcagc gccttctacc
     2401 agttctctgt caagcccacg gcctgacgcc cgagcaagtt 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 taatgggggc ggcaggccgg cgctggagag cattgttgcc cagttatctc gccctgatcc
     2821 ggcgttggcc gcgttgacca acgaccacct cgtcgccttg gcctgcctcg gcggacgtcc
     2881 tgcgctggat gcagtgaaaa agggattggg aggctcccca accccctacc ctaactatgc
     2941 caatgcagga cacgtggagg gacagagcgc cctgttcatg cgggataacg gcatctccga
     3001 gggcctggtg ttccacaaca atcccgaggg cacctgcggc ttttgcgtga acatgaccga
     3061 gacactgctg cccgagaatg ccaagatgac agtggtgcca cctgagggag caatccctgt
     3121 gaagagaggc gccaccggcg agacaaaggt gtttaccggc aactctaata gccctaagtc
     3181 cccaacaaag ggcggctgct ctggaggcag caccaacctg tctgacatca tcgagaagga
     3241 gacaggcaag cagctggtca tccaggagag catcctgatg ctgcccgaag aagtcgaaga
     3301 agtgatcgga aacaagcctg agagcgatat cctggtccat accgcctacg acgagagtac
     3361 cgacgaaaat gtgatgctgc tgacatccga cgccccagag tataagccct gggctctggt
     3421 catccaggat tccaacggag agaacaaaat caaaatgctg taatctagag ggcccgttta
     3481 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3541 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3601 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3661 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3721 tatggctcga gggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga
     3781 cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gactcgcaca
     3841 ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg
     3901 gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg
     3961 acgtgacaaa cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga
     4021 gcaatggcag cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc
     4081 gccgagagca gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg
     4141 gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca
     4201 gtcggctccc tcgttgaccg aatcaccgac ctctctcccc agggggatcc accggagctt
     4261 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     4321 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     4381 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     4441 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     4501 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     4561 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     4621 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     4681 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     4741 ctggaaacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     4801 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     4861 tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg
     4921 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tctagagtcg
     4981 gggcggccgg ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac
     5041 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     5101 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     5161 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     5221 taaaatcgat aaggatccgt cgaccgatgc ccttgagagc cttcaaccca gtcagctcct
     5281 tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc
     5341 aactcgtagg acaggtgccg gcagcgctct tccgcttcct cgctcactga ctcgctgcgc
     5401 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5461 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5521 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5581 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5641 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5701 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5761 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5821 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5881 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5941 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6001 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6061 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6121 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6181 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6241 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6301 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6361 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6421 tctggcccca gtgctgcaat gataccgcgg gacccacgct caccggctcc agatttatca
     6481 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6541 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6601 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6661 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6721 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6781 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6841 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6901 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6961 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7021 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7081 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7141 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7201 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7261 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
//
