LOCUS       sequence-342555-        6263 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 6263)
  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 6263)
  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(G1397C)-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..6263
                     /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             2175..2426
                     /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(2458..2475)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2464..2688
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2695..3205
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3124..3142
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3227..3826
                     /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(3227..3246)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3723..3743
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3973..4094
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3984..4003)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4038..4057
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4342..4359)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4513..5101)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4593..4612)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5272..6063,6064..6132))
                     /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     5895..5914
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6133..6237)
                     /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 ccgcaatccc tgtgaagaga ggcgccaccg
     2101 gcgagacaaa ggtgtttacc ggcaactcta atagccctaa gtccccaaca aagggcggct
     2161 gctctggagg cagcaccaac ctgtctgaca tcatcgagaa ggagacaggc aagcagctgg
     2221 tcatccagga gagcatcctg atgctgcccg aagaagtcga agaagtgatc ggaaacaagc
     2281 ctgagagcga tatcctggtc cataccgcct acgacgagag taccgacgaa aatgtgatgc
     2341 tgctgacatc cgacgcccca gagtataagc cctgggctct ggtcatccag gattccaacg
     2401 gagagaacaa aatcaaaatg ctgtaatcta gagggcccgt ttaaacccgc tgatcagcct
     2461 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2521 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     2581 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     2641 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct cgagggggtt
     2701 ggggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg
     2761 tggttccggg aaacgcagcg gcgccgaccc tgggactcgc acattcttca cgtccgttcg
     2821 cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc
     2881 gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc
     2941 cgcacgtctc actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg
     3001 accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg
     3061 ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc
     3121 gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga
     3181 ccgaatcacc gacctctctc cccaggggga tccaccggag cttaccatga ccgagtacaa
     3241 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
     3301 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
     3361 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     3421 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     3481 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     3541 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     3601 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     3661 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaaa cctccgcgcc
     3721 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     3781 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga
     3841 cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca tcggtacctt taagaccaat
     3901 gacttacaag gcagctgtag atcttagcca ctttctagag tcggggcggc cggccgcttc
     3961 gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     4021 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     4081 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     4141 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc
     4201 cgtcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     4261 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     4321 ccggcagcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4381 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4441 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4501 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4561 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4621 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4681 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4741 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4801 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4861 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4921 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4981 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5041 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5101 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5161 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5221 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5281 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5341 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5401 aatgataccg cgggacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5461 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5521 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5581 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5641 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5701 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5761 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5821 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5881 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5941 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6001 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6061 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6121 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6181 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6241 atttccccga aaagtgccac ctg
//
