LOCUS       sequence-391824-        7456 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Employ DdCBE-mediated mtDNA editing to ablate mtProtein(ND2) 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 7456)
  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 7456)
  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-ND2-W30-R18-G1333N-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7456
                     /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             3368..3619
                     /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(3651..3668)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3657..3881
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3888..4398
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4317..4335
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4420..5019
                     /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(4420..4439)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4916..4936
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5166..5287
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5177..5196)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5231..5250
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5535..5552)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5706..6294)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5786..5805)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6465..7256,7257..7325))
                     /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     7088..7107
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7326..7430)
                     /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 ctagtaatat
     1441 tggtggcaaa caggctcttg agacggttca gcgcctcctt ccagttctct gtcaagccca
     1501 cggactcacc ccagatcagg ttgtagcgat tgctagtaat attggtggca aacaggctct
     1561 tgagacggtt cagcgcctcc ttccagttct ctgtcaagcc cacggactca ccccagatca
     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 ctagtcatga cggtggcaaa caggctcttg aaaccgtcca
     1981 acgccttcta ccagttctct gtcaagccca cggactaacc ccagcgcaag ttgtagcgat
     2041 tgctagtaat gggggtggca aacaggctct tgagactgtt cagcgccttc taccagttct
     2101 ctgtcaagcc cacggcctga cgcccgagca agttgtagca attgctagta atgggggtgg
     2161 caaacaggct cttgagactg ttcagcgcct tctaccagtt ctctgtcaag cccacggcct
     2221 gacgcccgag caagttgtag cgattgctag taatattggt ggcaaacagg ctcttgagac
     2281 ggttcagcgc ctccttccag ttctctgtca agcccacgga ctcaccccag atcaagttgt
     2341 agcgattgct agtaatgggg gtggcaaaca ggctcttgag actgttcagc gccttctacc
     2401 agttctctgt caagcccacg gcctgacgcc cgagcaagtt 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 attgctagtc atgacggtgg caaacaggct cttgaaaccg tccaacgcct
     2701 tctaccagtt ctctgtcaag cccacggact aaccccagcg 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 cttccaatat tggtggcaaa caggctcttg agacggttca
     3001 gcgcctcctt ccagttctct gtcaagccca cggactcacc ccagatcaag ttgtagcgat
     3061 tgcctctaat gggggcggca ggccggcgct ggagagcatt gttgcccagt tatctcgccc
     3121 tgatccggcg ttggccgcgt tgaccaacga ccacctcgtc gccttggcct gcctcggcgg
     3181 acgtcctgcg ctggatgcag tgaaaaaggg attgggaggc tccggctcct acgccctggg
     3241 cccatatcag atctctgccc cacagctgcc agcatacaac ggacagaccg tgggcacatt
     3301 ctactatgtg aatgacgccg gcggcctgga gtctaaggtg tttagctccg gcggctctgg
     3361 aggcagcacc aacctgtctg acatcatcga gaaggagaca ggcaagcagc tggtcatcca
     3421 ggagagcatc ctgatgctgc ccgaagaagt cgaagaagtg atcggaaaca agcctgagag
     3481 cgatatcctg gtccataccg cctacgacga gagtaccgac gaaaatgtga tgctgctgac
     3541 atccgacgcc ccagagtata agccctgggc tctggtcatc caggattcca acggagagaa
     3601 caaaatcaaa atgctgtaat ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     3661 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3721 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3781 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3841 agacaatagc aggcatgctg gggatgcggt gggctctatg gctcgagggg gttggggttg
     3901 cgccttttcc aaggcagccc tgggtttgcg cagggacgcg gctgctctgg gcgtggttcc
     3961 gggaaacgca gcggcgccga ccctgggact cgcacattct tcacgtccgt tcgcagcgtc
     4021 acccggatct tcgccgctac ccttgtgggc cccccggcga cgcttcctgc tccgccccta
     4081 agtcgggaag gttccttgcg gttcgcggcg tgccggacgt gacaaacgga agccgcacgt
     4141 ctcactagta ccctcgcaga cggacagcgc cagggagcaa tggcagcgcg ccgaccgcga
     4201 tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg agagcagcgg ccgggaaggg
     4261 gcggtgcggg aggcggggtg tggggcggta gtgtgggccc tgttcctgcc cgcgcggtgt
     4321 tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg gctccctcgt tgaccgaatc
     4381 accgacctct ctccccaggg ggatccaccg gagcttacca tgaccgagta caagcccacg
     4441 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     4501 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     4561 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     4621 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     4681 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     4741 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     4801 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     4861 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg aaacctccgc gccccgcaac
     4921 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     4981 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag
     5041 cgcccgaccg aaaggagcgc acgaccccat gcatcggtac ctttaagacc aatgacttac
     5101 aaggcagctg tagatcttag ccactttcta gagtcggggc ggccggccgc ttcgagcaga
     5161 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     5221 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     5281 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     5341 ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgtcgac
     5401 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     5461 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
     5521 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5581 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5641 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5701 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5761 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5821 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5881 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5941 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6001 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6061 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6121 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6181 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6241 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6301 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6361 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6421 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6481 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6541 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6601 ccgcgggacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6661 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6721 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6781 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6841 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6901 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6961 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7021 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7081 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7141 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7201 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7261 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7321 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7381 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7441 cgaaaagtgc cacctg
//
