LOCUS       sequence-391810-        7048 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Employ DdCBE-mediated mtDNA editing to ablate mtProtein(CYTB) 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 7048)
  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 7048)
  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-CYTB-W77-R14-G1333N-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7048
                     /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             2960..3211
                     /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(3243..3260)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3249..3473
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3480..3990
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3909..3927
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4012..4611
                     /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(4012..4031)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4508..4528
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4758..4879
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4769..4788)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4823..4842
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5127..5144)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5298..5886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5378..5397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6057..6848,6849..6917))
                     /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     6680..6699
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6918..7022)
                     /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 agtaatgggg gtggcaaaca ggctcttgag actgttcagc gccttctacc
     1381 agttctctgt caagcccacg gcctgacgcc cgagcaagtt gtagcgattg ctagtaacaa
     1441 tggtggcaaa caggctcttg aaaccgtaca gcgcctactg ccagttctct gtcaagccca
     1501 cggtctgact ccggagcagg ttgtagcgat tgctagtaac aatggtggca aacaggctct
     1561 tgaaaccgta cagcgcctac tgccagttct ctgtcaagcc cacggtctga ctccggagca
     1621 agttgtagcg attgctagtc atgacggtgg caaacaggct cttgaaaccg tccaacgcct
     1681 tctaccagtt ctctgtcaag cccacggact aaccccagcg caagtggtag cgattgctag
     1741 taacaatggt ggcaaacagg ctcttgaaac cgtacagcgc ctactgccag ttctctgtca
     1801 agcccacggt ctgactccgg agcaagttgt agcgattgct agtaatgggg gtggcaaaca
     1861 ggctcttgag actgttcagc gccttctacc agttctctgt caagcccacg gcctgacgcc
     1921 cgagcaagtt gtagcgattg ctagtaacaa tggtggcaaa caggctcttg aaaccgtaca
     1981 gcgcctactg ccagttctct gtcaagccca cggtctgact ccggagcaag 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 taacaatggt ggcaaacagg ctcttgaaac
     2281 cgtacagcgc ctactgccag ttctctgtca agcccacggt ctgactccgg agcaagttgt
     2341 agcgattgct agtaacaatg gtggcaaaca ggctcttgaa accgtacagc gcctactgcc
     2401 agttctctgt caagcccacg gtctgactcc ggagcaagtt gtagcgattg ctagtaatgg
     2461 gggtggcaaa caggctcttg agactgttca gcgccttcta ccagttctct gtcaagccca
     2521 cggcctgacg cccgagcaag ttgtagcgat agccagtaat attggtggca aacaggctct
     2581 tgagacggtt cagcgcctcc ttccagttct ctgtcaagcc cacggactca ccccagatca
     2641 agttgtagcg attgcaagta atgggggcgg caggccggcg ctggagagca ttgttgccca
     2701 gttatctcgc cctgatccgg cgttggccgc gttgaccaac gaccacctcg tcgccttggc
     2761 ctgcctcggc ggacgtcctg cgctggatgc agtgaaaaag ggattgggag gctccggctc
     2821 ctacgccctg ggcccatatc agatctctgc cccacagctg ccagcataca acggacagac
     2881 cgtgggcaca ttctactatg tgaatgacgc cggcggcctg gagtctaagg tgtttagctc
     2941 cggcggctct ggaggcagca ccaacctgtc tgacatcatc gagaaggaga caggcaagca
     3001 gctggtcatc caggagagca tcctgatgct gcccgaagaa gtcgaagaag tgatcggaaa
     3061 caagcctgag agcgatatcc tggtccatac cgcctacgac gagagtaccg acgaaaatgt
     3121 gatgctgctg acatccgacg ccccagagta taagccctgg gctctggtca tccaggattc
     3181 caacggagag aacaaaatca aaatgctgta atctagaggg cccgtttaaa cccgctgatc
     3241 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3301 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3361 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3421 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggctcgagg
     3481 gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct
     3541 gggcgtggtt ccgggaaacg cagcggcgcc gaccctggga ctcgcacatt cttcacgtcc
     3601 gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct
     3661 gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg
     3721 gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg
     3781 cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc
     3841 ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg
     3901 cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc
     3961 gttgaccgaa tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag
     4021 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     4081 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     4141 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     4201 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     4261 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     4321 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     4381 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     4441 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggaaacctcc
     4501 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     4561 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc
     4621 cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga
     4681 ccaatgactt acaaggcagc tgtagatctt agccactttc tagagtcggg gcggccggcc
     4741 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     4801 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     4861 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     4921 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa
     4981 ggatccgtcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
     5041 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
     5101 aggtgccggc agcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5161 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5221 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5281 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5341 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5401 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5461 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5521 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5581 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5641 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5701 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5761 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5821 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5881 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5941 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6001 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6061 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6121 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6181 gctgcaatga taccgcggga cccacgctca ccggctccag atttatcagc aataaaccag
     6241 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6301 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6361 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6421 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6481 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6541 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6601 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6661 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6721 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6781 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6841 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6901 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6961 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7021 cgcacatttc cccgaaaagt gccacctg
//
