LOCUS       sequence-416837-        7522 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 7522)
  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 7522)
  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-L17-FZY2-S100N-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..7522
                     /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             3434..3685
                     /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(3717..3734)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3723..3947
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3954..4464
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4383..4401
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4486..5085
                     /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(4486..4505)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4982..5002
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5232..5353
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5243..5262)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5297..5316
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5601..5618)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5772..6360)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5852..5871)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6531..7322,7323..7391))
                     /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     7154..7173
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7392..7496)
                     /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 ctagtcatga
     1441 cggtggcaaa caggctcttg aaaccgtcca acgccttcta ccagttctct gtcaagccca
     1501 cggactaacc ccagcgcagg 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 taatgggggt ggcaaacagg ctcttgagac tgttcagcgc cttctaccag ttctctgtca
     1801 agcccacggc ctgacgcccg agcaagttgt 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 attggtggca aacaggctct tgagacggtt cagcgcctcc ttccagttct
     2101 ctgtcaagcc cacggactca ccccagatca agttgtagca attgctagta atgggggtgg
     2161 caaacaggct cttgagactg ttcagcgcct tctaccagtt ctctgtcaag cccacggcct
     2221 gacgcccgag caagttgtag cgattgctag taatgggggt ggcaaacagg ctcttgagac
     2281 tgttcagcgc cttctaccag ttctctgtca agcccacggc ctgacgcccg agcaagttgt
     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 agccagtaat attggtggca aacaggctct
     2581 tgagacggtt cagcgcctcc ttccagttct ctgtcaagcc cacggactca ccccagatca
     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 agtcatgacg gtggcaaaca
     2881 ggctcttgaa accgtccaac gccttctacc agttctctgt caagcccacg gactaacccc
     2941 agcgcaagtt gtagcgattg cttccaatat tggcggcagg ccggcgctgg agagcattgt
     3001 tgcccagtta tctcgccctg atccggcgtt ggccgcgttg accaacgacc acctcgtcgc
     3061 gctagcctgc ctcggcggac gtcctgcgct ggatgcagtg aaaaagggat tgggaggctc
     3121 catgagcttg ccagagtacg atggcaccac cacccatggc gtgttggtgt tggatgatgg
     3181 cacccagatc ggcttcacca gcggcaacgg cgatccaagg tacaccaact acaggaacaa
     3241 cggccacgtg gaacaaaaga gcgcattgta catgagggag aacaacatca gcaacgcaac
     3301 cgtgtaccat aacaacacca acggcacctg cggctactgc aacaccatga ccgcaacctt
     3361 tttgccagag ggcgcaacct tgaccgtggt gccaccagag aacgcagtgg caaacaacag
     3421 ctctggaggc agcaccaacc tgtctgacat catcgagaag gagacaggca agcagctggt
     3481 catccaggag agcatcctga tgctgcccga agaagtcgaa gaagtgatcg gaaacaagcc
     3541 tgagagcgat atcctggtcc ataccgccta cgacgagagt accgacgaaa atgtgatgct
     3601 gctgacatcc gacgccccag agtataagcc ctgggctctg gtcatccagg attccaacgg
     3661 agagaacaaa atcaaaatgc tgtaatctag agggcccgtt taaacccgct gatcagcctc
     3721 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3781 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3841 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3901 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctc gagggggttg
     3961 gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt
     4021 ggttccggga aacgcagcgg cgccgaccct gggactcgca cattcttcac gtccgttcgc
     4081 agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg
     4141 cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc
     4201 gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga
     4261 ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg
     4321 gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg
     4381 cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac
     4441 cgaatcaccg acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag
     4501 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     4561 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     4621 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     4681 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     4741 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     4801 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     4861 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     4921 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggaaac ctccgcgccc
     4981 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     5041 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacgccc gccccacgac
     5101 ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat cggtaccttt aagaccaatg
     5161 acttacaagg cagctgtaga tcttagccac tttctagagt cggggcggcc ggccgcttcg
     5221 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     5281 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     5341 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     5401 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc
     5461 gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     5521 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     5581 cggcagcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5641 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5701 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5761 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5821 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5881 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5941 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6001 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6061 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6121 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6181 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6241 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6301 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6361 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6421 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6481 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6541 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6601 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6661 atgataccgc gggacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6721 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6781 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6841 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6901 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6961 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     7021 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     7081 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7141 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7201 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7261 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7321 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7381 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7441 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7501 tttccccgaa aagtgccacc tg
//
