LOCUS       sequence-391301-        7150 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 7150)
  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 7150)
  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-R15-G1333N-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7150
                     /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             3062..3313
                     /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(3345..3362)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3351..3575
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3582..4092
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4011..4029
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4114..4713
                     /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(4114..4133)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4610..4630
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4860..4981
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4871..4890)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4925..4944
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5229..5246)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5400..5988)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5480..5499)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6159..6950,6951..7019))
                     /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     6782..6801
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7020..7124)
                     /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 tgctagtaat gggggtggca aacaggctct
     1561 tgagactgtt cagcgccttc taccagttct ctgtcaagcc cacggcctga cgcccgagca
     1621 agttgtagcg attgctagta atattggtgg caaacaggct cttgagacgg ttcagcgcct
     1681 ccttccagtt ctctgtcaag cccacggact caccccagat caagtggtag cgattgctag
     1741 taacaatggt ggcaaacagg ctcttgaaac cgtacagcgc ctactgccag ttctctgtca
     1801 agcccacggt ctgactccgg agcaagttgt agcgattgct agtaacaatg gtggcaaaca
     1861 ggctcttgaa accgtacagc gcctactgcc agttctctgt caagcccacg gtctgactcc
     1921 ggagcaagtt gtagcgattg ctagtaacaa tggtggcaaa caggctcttg aaaccgtaca
     1981 gcgcctactg ccagttctct gtcaagccca cggtctgact ccggagcaag ttgtagcgat
     2041 tgctagtcat gacggtggca aacaggctct tgaaaccgtc caacgccttc taccagttct
     2101 ctgtcaagcc cacggactaa ccccagcgca agttgtagca attgctagta atgggggtgg
     2161 caaacaggct cttgagactg ttcagcgcct tctaccagtt ctctgtcaag cccacggcct
     2221 gacgcccgag 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 ctagtaatgg
     2461 gggtggcaaa caggctcttg agactgttca gcgccttcta ccagttctct gtcaagccca
     2521 cggcctgacg cccgagcaag ttgtagcgat agccagtaac aatggtggca aacaggctct
     2581 tgaaaccgta cagcgcctac tgccagttct ctgtcaagcc cacggtctga ctccggagca
     2641 agttgtagcg attgctagta atgggggtgg caaacaggct cttgagactg ttcagcgcct
     2701 tctaccagtt ctctgtcaag cccacggcct gacgcccgag caagttgtag cgattgcaag
     2761 taatgggggc ggcaggccgg cgctggagag cattgttgcc cagttatctc gccctgatcc
     2821 ggcgttggcc gcgttgacca acgaccacct cgtcgccttg gcctgcctcg gcggacgtcc
     2881 tgcgctggat gcagtgaaaa agggattggg aggctccggc tcctacgccc tgggcccata
     2941 tcagatctct gccccacagc tgccagcata caacggacag accgtgggca cattctacta
     3001 tgtgaatgac gccggcggcc tggagtctaa ggtgtttagc tccggcggct ctggaggcag
     3061 caccaacctg tctgacatca tcgagaagga gacaggcaag cagctggtca tccaggagag
     3121 catcctgatg ctgcccgaag aagtcgaaga agtgatcgga aacaagcctg agagcgatat
     3181 cctggtccat accgcctacg acgagagtac cgacgaaaat gtgatgctgc tgacatccga
     3241 cgccccagag tataagccct gggctctggt catccaggat tccaacggag agaacaaaat
     3301 caaaatgctg taatctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     3361 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3421 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3481 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3541 tagcaggcat gctggggatg cggtgggctc tatggctcga gggggttggg gttgcgcctt
     3601 ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa
     3661 cgcagcggcg ccgaccctgg gactcgcaca ttcttcacgt ccgttcgcag cgtcacccgg
     3721 atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg
     3781 gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact
     3841 agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct
     3901 gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg
     3961 cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca
     4021 ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac
     4081 ctctctcccc agggggatcc accggagctt accatgaccg agtacaagcc cacggtgcgc
     4141 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     4201 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     4261 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     4321 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     4381 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     4441 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     4501 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     4561 gcggccgagc gcgccggggt gcccgccttc ctggaaacct ccgcgccccg caacctcccc
     4621 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     4681 acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg
     4741 accgaaagga gcgcacgacc ccatgcatcg gtacctttaa gaccaatgac ttacaaggca
     4801 gctgtagatc ttagccactt tctagagtcg gggcggccgg ccgcttcgag cagacatgat
     4861 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     4921 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     4981 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     5041 ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatccgt cgaccgatgc
     5101 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
     5161 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     5221 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5281 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5341 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5401 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5461 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5521 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5581 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5641 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5701 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5761 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5821 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5881 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5941 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6001 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6061 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6121 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6181 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6241 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcgg
     6301 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6361 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6421 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6481 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6541 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6601 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6661 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6721 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6781 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6841 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6901 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6961 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7021 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7081 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7141 gtgccacctg
//
