LOCUS       sequence-391812-        7414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Employ DdCBE-mediated mtDNA editing to ablate mtProtein(ND3) by 
            swapping Gln codon CAA with stop codon TAA in C6 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7414)
  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 7414)
  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-ND3-Q26-L18-G1397C-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7414
                     /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             3326..3577
                     /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(3609..3626)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3615..3839
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3846..4356
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4275..4293
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4378..4977
                     /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(4378..4397)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4874..4894
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5124..5245
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5135..5154)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5189..5208
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5493..5510)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5664..6252)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5744..5763)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6423..7214,7215..7283))
                     /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     7046..7065
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7284..7388)
                     /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 ctagtcatga
     1441 cggtggcaaa caggctcttg aaaccgtcca acgccttcta ccagttctct gtcaagccca
     1501 cggactaacc ccagcgcagg ttgtagcgat tgctagtaat gggggtggca aacaggctct
     1561 tgagactgtt cagcgccttc taccagttct ctgtcaagcc cacggcctga cgcccgagca
     1621 agttgtagcg attgctagtc atgacggtgg caaacaggct cttgaaaccg tccaacgcct
     1681 tctaccagtt ctctgtcaag cccacggact aaccccagcg caagtggtag cgattgctag
     1741 taatattggt ggcaaacagg ctcttgagac ggttcagcgc ctccttccag ttctctgtca
     1801 agcccacgga ctcaccccag atcaagttgt agcgattgct agtaatgggg gtggcaaaca
     1861 ggctcttgag actgttcagc gccttctacc agttctctgt caagcccacg gcctgacgcc
     1921 cgagcaagtt gtagcgattg ctagtaatgg gggtggcaaa caggctcttg agactgttca
     1981 gcgccttcta ccagttctct gtcaagccca cggcctgacg cccgagcaag ttgtagcgat
     2041 tgctagtaat gggggtggca aacaggctct tgagactgtt cagcgccttc taccagttct
     2101 ctgtcaagcc cacggcctga cgcccgagca agttgtagca attgctagtc atgacggtgg
     2161 caaacaggct cttgaaaccg tccaacgcct tctaccagtt ctctgtcaag cccacggact
     2221 aaccccagcg caagttgtag cgattgctag taatattggt ggcaaacagg ctcttgagac
     2281 ggttcagcgc ctccttccag ttctctgtca agcccacgga ctcaccccag atcaagttgt
     2341 agcgattgct agtaatattg gtggcaaaca ggctcttgag acggttcagc gcctccttcc
     2401 agttctctgt caagcccacg gactcacccc agatcaagtt 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 acaatggtgg caaacaggct cttgaaaccg tacagcgcct
     2701 actgccagtt ctctgtcaag cccacggtct gactccggag caagttgtag cgattgcaag
     2761 tcatgacggt ggcaaacagg ctcttgaaac cgtccaacgc cttctaccag ttctctgtca
     2821 agcccacgga ctaaccccag cgcaagttgt agcgattgct agtaatattg gtggcaaaca
     2881 ggctcttgag acggttcagc gcctccttcc agttctctgt caagcccacg gactcacccc
     2941 agatcaagtt gtagcgattg cttccaatgg gggtggcaaa caggctcttg agactgttca
     3001 gcgccttcta ccagttctct gtcaagccca cggcctgacg cccgagcaag ttgtagcgat
     3061 tgcctctaat gggggcggca ggccggcgct ggagagcatt gttgcccagt tatctcgccc
     3121 tgatccggcg ttggccgcgt tgaccaacga ccacctcgtc gccttggcct gcctcggcgg
     3181 acgtcctgcg ctggatgcag tgaaaaaggg attgggaggc tccgcaatcc ctgtgaagag
     3241 aggcgccacc ggcgagacaa aggtgtttac cggcaactct aatagcccta agtccccaac
     3301 aaagggcggc tgctctggag gcagcaccaa cctgtctgac atcatcgaga aggagacagg
     3361 caagcagctg gtcatccagg agagcatcct gatgctgccc gaagaagtcg aagaagtgat
     3421 cggaaacaag cctgagagcg atatcctggt ccataccgcc tacgacgaga gtaccgacga
     3481 aaatgtgatg ctgctgacat ccgacgcccc agagtataag ccctgggctc tggtcatcca
     3541 ggattccaac ggagagaaca aaatcaaaat gctgtaatct agagggcccg tttaaacccg
     3601 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3661 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3721 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3781 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3841 tcgagggggt tggggttgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc
     3901 tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggactcg cacattcttc
     3961 acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg
     4021 cttcctgctc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga
     4081 caaacggaag ccgcacgtct cactagtacc ctcgcagacg gacagcgcca gggagcaatg
     4141 gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcagg gcgcgccgag
     4201 agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg
     4261 ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc
     4321 tccctcgttg accgaatcac cgacctctct ccccaggggg atccaccgga gcttaccatg
     4381 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     4441 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     4501 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     4561 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     4621 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     4681 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     4741 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     4801 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggaa
     4861 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     4921 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc
     4981 ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgc atcggtacct
     5041 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttctaga gtcggggcgg
     5101 ccggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     5161 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     5221 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     5281 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat
     5341 cgataaggat ccgtcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     5401 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     5461 taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5521 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5581 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5641 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5701 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5761 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5821 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5881 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5941 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6001 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6061 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6121 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6181 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6241 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6301 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6361 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6421 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6481 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6541 cccagtgctg caatgatacc gcgggaccca cgctcaccgg ctccagattt atcagcaata
     6601 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6661 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6721 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6781 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6841 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6901 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6961 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7021 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7081 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7141 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7201 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7261 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7321 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7381 gttccgcgca catttccccg aaaagtgcca cctg
//
