LOCUS       sequence-342557-        6497 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  One-step assembly of custom RVD arrays to construct DdCBE, TALE, 
            etc..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6497)
  AUTHORS   Guo J, Zhang X, Chen X, Sun H, Dai Y, Wang J, Qian X, Tan L, Lou X, 
            Shen B
  TITLE     Precision modeling of mitochondrial diseases in zebrafish via 
            DdCBE-mediated mtDNA base editing.
  JOURNAL   Cell Discov. 2021 Sep 3;7(1):78. doi: 10.1038/s41421-021-00307-9.
  PUBMED    34480028
REFERENCE   2  (bases 1 to 6497)
  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: "Cell 
            Discov."; date: "2021-09-3"; volume: "7(1):78. doi"; pages: " 
            10.1038/s41421-021-00307-9"
COMMENT     Sequence Label: pGL3-MTS-ccdb-DddA(G1397N)-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..6497
                     /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             2409..2660
                     /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(2692..2709)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2698..2922
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2929..3439
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3358..3376
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3461..4060
                     /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(3461..3480)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3957..3977
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4207..4328
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4218..4237)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4272..4291
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4576..4593)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4747..5335)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4827..4846)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5506..6297,6298..6366))
                     /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     6129..6148
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6367..6471)
                     /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 ccggctccta cgccctgggc ccatatcaga
     2101 tctctgcccc acagctgcca gcatacaacg gacagaccgt gggcacattc tactatgtga
     2161 atgacgccgg cggcctggag tctaaggtgt ttagctccgg cggcccaacc ccctacccta
     2221 actatgccaa tgcaggacac gtggagggac agagcgccct gttcatgcgg gataacggca
     2281 tctccgaggg cctggtgttc cacaacaatc ccgagggcac ctgcggcttt tgcgtgaaca
     2341 tgaccgagac actgctgccc gagaatgcca agatgacagt ggtgccacct gagggatctg
     2401 gaggcagcac caacctgtct gacatcatcg agaaggagac aggcaagcag ctggtcatcc
     2461 aggagagcat cctgatgctg cccgaagaag tcgaagaagt gatcggaaac aagcctgaga
     2521 gcgatatcct ggtccatacc gcctacgacg agagtaccga cgaaaatgtg atgctgctga
     2581 catccgacgc cccagagtat aagccctggg ctctggtcat ccaggattcc aacggagaga
     2641 acaaaatcaa aatgctgtaa tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     2701 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2761 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2821 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2881 aagacaatag caggcatgct ggggatgcgg tgggctctat ggctcgaggg ggttggggtt
     2941 gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc
     3001 cgggaaacgc agcggcgccg accctgggac tcgcacattc ttcacgtccg ttcgcagcgt
     3061 cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct
     3121 aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg
     3181 tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg
     3241 atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg
     3301 ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg
     3361 ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat
     3421 caccgacctc tctccccagg gggatccacc ggagcttacc atgaccgagt acaagcccac
     3481 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     3541 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     3601 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     3661 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     3721 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     3781 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     3841 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     3901 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gaaacctccg cgccccgcaa
     3961 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     4021 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca
     4081 gcgcccgacc gaaaggagcg cacgacccca tgcatcggta cctttaagac caatgactta
     4141 caaggcagct gtagatctta gccactttct agagtcgggg cggccggccg cttcgagcag
     4201 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     4261 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     4321 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     4381 aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatccgtcga
     4441 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     4501 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     4561 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4621 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4681 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4741 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4801 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4861 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4921 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4981 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5041 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5101 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5161 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5221 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5281 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5341 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5401 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5461 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5521 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5581 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5641 accgcgggac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5701 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5761 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5821 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5881 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5941 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6001 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6061 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6121 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6181 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6241 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6301 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6361 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6421 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6481 ccgaaaagtg ccacctg
//
