LOCUS       sequence-405612-        6522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A backbone containing a RsDddA fragment D for constructing 
            mitochondria-targeted RsDdCBE expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6522)
  AUTHORS   Cheng K, Li C, Jin J, Qian X, Guo J, Shen L, Dai Y, Zhang X, Li Z, 
            Guan Y, Zhou F, Tang J, Zhang J, Shen B, Lou X
  TITLE     Engineering RsDddA as mitochondrial base editor with wide target 
            compatibility and enhanced activity.
  JOURNAL   Mol Ther Nucleic Acids. 2023 Sep 9;34:102028. doi: 
            10.1016/j.omtn.2023.09.005. eCollection 2023 Dec 12.
  PUBMED    37744175
REFERENCE   2  (bases 1 to 6522)
  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: "Mol Ther 
            Nucleic Acids."; date: "2023-09-9"; volume: "34:102028. doi"; pages:
            " 10.1016/j.omtn.2023.09.005. eCollection 2023 Dec 12"
COMMENT     Sequence Label: pGL3-MTS-CCDB-N136-RsDddA-D-UGI-PURO
FEATURES             Location/Qualifiers
     source          1..6522
                     /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             1645..1950
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1829..1848
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2430..2684
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLA"
     primer_bind     complement(2717..2734)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2723..2947
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2954..3464
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3383..3401
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3486..4085
                     /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(3486..3505)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3982..4002
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4232..4353
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4243..4262)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4297..4316
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4601..4618)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4772..5360)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4852..4871)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5531..6322,6323..6391))
                     /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     6154..6173
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6392..6496)
                     /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 ctgaccccag agaccggctt
     1321 actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata
     1381 tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca
     1441 gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg
     1501 gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag
     1561 cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg
     1621 ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc
     1681 cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg
     1741 gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg
     1801 gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg
     1861 gtgtccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac
     1921 gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca cagccagggt
     1981 ctctcgcgtt gaccaacgac cacctcgtcg ccttggcctg cctcggcgga cgtcctgcgc
     2041 tggatgcagt gaaaaaggga ttgggaggct ccgctagcaa gtgcagactg ctggtgcaca
     2101 acgccaactg caaccaggag aagcccgtgc tgcccaagta cgacggcaag accaccgagg
     2161 gcgtgatggt gacccccgac ggcaagcaga tcagcttcaa gtccggcaac agcagcaccc
     2221 ccagctaccc ccagtacaag gcccagagcg ccagccacgt ggagggcaag gccgccctgt
     2281 acatgaggga gaacggcatc aacgaggcca ccgtgttcca caacaacccc aacggcacct
     2341 gcggcttctg cgacagacag gtgcccgccc tgctgcccaa gggcgccaag ctgaccgtgg
     2401 tgcccccctc caacgcatca ggcgggagca ccaacctgtc tgacatcatc gagaaggaga
     2461 caggcaagca gctggtcatc caggagagca tcctgatgct gcccgaagaa gtcgaagaag
     2521 tgatcggaaa caagcctgag agcgatatcc tggtccatac cgcctacgac gagagtaccg
     2581 acgaaaatgt gatgctgctg acatccgacg ccccagagta taagccctgg gctctggtca
     2641 tccaggattc caacggagag aacaaaatca aaatgctggc ttaagtctag agggcccgtt
     2701 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     2761 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     2821 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     2881 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     2941 tctatggctc gagggggttg gggttgcgcc ttttccaagg cagccctggg tttgcgcagg
     3001 gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct gggactcgca
     3061 cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc
     3121 cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc
     3181 ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga cagcgccagg
     3241 gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc
     3301 gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt
     3361 gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag cgcacgtcgg
     3421 cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccagggggat ccaccggagc
     3481 ttaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
     3541 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
     3601 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     3661 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     3721 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     3781 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     3841 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     3901 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     3961 tcctggaaac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     4021 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     4081 cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat
     4141 cggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttctagagt
     4201 cggggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc
     4261 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4321 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4381 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     4441 ggtaaaatcg ataaggatcc gtcgaccgat gcccttgaga gccttcaacc cagtcagctc
     4501 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     4561 gcaactcgta ggacaggtgc cggcagcgct cttccgcttc ctcgctcact gactcgctgc
     4621 gctcggtcgt taggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4681 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4741 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4801 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4861 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4921 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4981 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5041 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5101 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5161 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5221 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5281 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5341 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5401 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5461 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5521 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5581 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5641 catctggccc cagtgctgca atgataccgc gggacccacg ctcaccggct ccagatttat
     5701 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5761 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5821 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5881 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5941 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6001 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6061 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6121 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6181 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6241 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6301 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6361 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6421 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6481 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tg
//
