LOCUS       sequence-416863-        7420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To install m.G3635 mutation on mtDNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7420)
  AUTHORS   Sun H, Wang Z, Shen L, Feng Y, Han L, Qian X, Meng R, Ji K, Liang D,
            Zhou F, Lou X, Zhang J, Shen B
  TITLE     Developing mitochondrial base editors with diverse context 
            compatibility and high fidelity via saturated spacer library.
  JOURNAL   Nat Commun. 2023 Oct 19;14(1):6625. doi: 10.1038/s41467-023-42359-3.
  PUBMED    37857619
REFERENCE   2  (bases 1 to 7420)
  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: "Nat 
            Commun."; date: "2023-10-19"; volume: "14(1):6625. doi"; pages: " 
            10.1038/s41467-023-42359-3"
COMMENT     Sequence Label: pGL3-MTS-G3635A-R16-FZY2-S100N-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7420
                     /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             3332..3583
                     /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(3615..3632)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3621..3845
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3852..4362
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4281..4299
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4384..4983
                     /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(4384..4403)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4880..4900
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5130..5251
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5141..5160)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5195..5214
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5499..5516)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5670..6258)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5750..5769)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6429..7220,7221..7289))
                     /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     7052..7071
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7290..7394)
                     /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 ggcaaacggg gcgccggcgc
     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 ctagtaatat
     1441 tggtggcaaa caggctcttg agacggttca gcgcctcctt ccagttctct gtcaagccca
     1501 cggactcacc ccagatcagg ttgtagcgat tgctagtaac aatggtggca aacaggctct
     1561 tgaaaccgta cagcgcctac tgccagttct ctgtcaagcc cacggtctga ctccggagca
     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 ctagtaatat tggtggcaaa caggctcttg agacggttca
     1981 gcgcctcctt ccagttctct gtcaagccca cggactcacc ccagatcaag 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 taacaatggt ggcaaacagg ctcttgaaac
     2281 cgtacagcgc ctactgccag ttctctgtca agcccacggt ctgactccgg agcaagttgt
     2341 agcgattgct agtaatattg gtggcaaaca ggctcttgag acggttcagc gcctccttcc
     2401 agttctctgt caagcccacg gactcacccc agatcaagtt gtagcgattg ctagtaacaa
     2461 tggtggcaaa caggctcttg aaaccgtaca gcgcctactg ccagttctct gtcaagccca
     2521 cggtctgact ccggagcaag ttgtagcgat agccagtaat gggggtggca aacaggctct
     2581 tgagactgtt cagcgccttc taccagttct ctgtcaagcc cacggcctga cgcccgagca
     2641 agttgtagcg attgctagta atattggtgg caaacaggct cttgagacgg ttcagcgcct
     2701 ccttccagtt ctctgtcaag cccacggact caccccagat caagttgtag cgattgcaag
     2761 taatattggt ggcaaacagg ctcttgagac ggttcagcgc ctccttccag ttctctgtca
     2821 agcccacgga ctcaccccag atcaagttgt agcgattgct tccaatattg gcggcaggcc
     2881 ggcgctggag agcattgttg cccagttatc tcgccctgat ccggcgttgg ccgcgttgac
     2941 caacgaccac ctcgtcgcgc tagcctgcct cggcggacgt cctgcgctgg atgcagtgaa
     3001 aaagggattg ggaggctcca tgagcttgcc agagtacgat ggcaccacca cccatggcgt
     3061 gttggtgttg gatgatggca cccagatcgg cttcaccagc ggcaacggcg atccaaggta
     3121 caccaactac aggaacaacg gccacgtgga acaaaagagc gcattgtaca tgagggagaa
     3181 caacatcagc aacgcaaccg tgtaccataa caacaccaac ggcacctgcg gctactgcaa
     3241 caccatgacc gcaacctttt tgccagaggg cgcaaccttg accgtggtgc caccagagaa
     3301 cgcagtggca aacaacagct ctggaggcag caccaacctg tctgacatca tcgagaagga
     3361 gacaggcaag cagctggtca tccaggagag catcctgatg ctgcccgaag aagtcgaaga
     3421 agtgatcgga aacaagcctg agagcgatat cctggtccat accgcctacg acgagagtac
     3481 cgacgaaaat gtgatgctgc tgacatccga cgccccagag tataagccct gggctctggt
     3541 catccaggat tccaacggag agaacaaaat caaaatgctg taatctagag ggcccgttta
     3601 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3661 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3721 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3781 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3841 tatggctcga gggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga
     3901 cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gactcgcaca
     3961 ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg
     4021 gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg
     4081 acgtgacaaa cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga
     4141 gcaatggcag cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc
     4201 gccgagagca gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg
     4261 gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca
     4321 gtcggctccc tcgttgaccg aatcaccgac ctctctcccc agggggatcc accggagctt
     4381 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     4441 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     4501 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     4561 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     4621 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     4681 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     4741 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     4801 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     4861 ctggaaacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     4921 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     4981 tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg
     5041 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tctagagtcg
     5101 gggcggccgg ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac
     5161 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     5221 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     5281 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     5341 taaaatcgat aaggatccgt cgaccgatgc ccttgagagc cttcaaccca gtcagctcct
     5401 tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc
     5461 aactcgtagg acaggtgccg gcagcgctct tccgcttcct cgctcactga ctcgctgcgc
     5521 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5581 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5641 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5701 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5761 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5821 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5881 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5941 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6001 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6061 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6121 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6181 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6241 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6301 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6361 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6421 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6481 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6541 tctggcccca gtgctgcaat gataccgcgg gacccacgct caccggctcc agatttatca
     6601 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6661 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6721 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6781 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6841 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6901 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6961 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7021 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7081 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7141 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7201 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7261 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7321 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7381 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
//
