LOCUS       sequence-343235-        6143 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 6143)
  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 6143)
  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-NLS-ccdb-DddA(G1397C)-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..6143
                     /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             680..700
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             701..766
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1525..1830
                     /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     1709..1728
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2055..2306
                     /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(2338..2355)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2344..2568
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2575..3085
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3004..3022
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3107..3706
                     /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(3107..3126)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3603..3623
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3853..3974
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3864..3883)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3918..3937
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4222..4239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4393..4981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4473..4492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5152..5943,5944..6012))
                     /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     5775..5794
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6013..6117)
                     /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 accatggctc caaagaagaa gcgtaaggta gactacaaag accatgacgg
      721 tgattataaa gatcatgaca tcgattacaa ggatgacgat gacaagggta ccgtggatct
      781 acgcacgctc ggctacagcc agcagcaaca ggagaagatc aaaccgaagg ttcgttcgac
      841 agtggcgcag caccacgagg cactggtcgg ccatgggttt acacacgcgc acatcgttgc
      901 gctcagccaa cacccggcag cgttagggac cgtcgctgtc aagtatcagg acatgatcgc
      961 agcgttgcca gaggcgacac acgaagcgat cgttggcgtc ggcaaacagt ggtccggcgc
     1021 acgcgctctg gaggccttgc tcacggtggc gggagagttg agaggtccac cgttacagtt
     1081 ggacacaggc caacttctca agattgcaaa acgtggcggc gtgaccgcag tggaggcagt
     1141 gcatgcatgg cgcaatgcac tgacgggtgc ccccctgaac ctgaccccag agaccggctt
     1201 actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata
     1261 tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca
     1321 gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg
     1381 gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag
     1441 cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg
     1501 ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc
     1561 cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg
     1621 gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg
     1681 gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg
     1741 gtgtccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac
     1801 gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca cagccagggt
     1861 ctctcgcgtt gaccaacgac cacctcgtcg ccttggcctg cctcggcgga cgtcctgcgc
     1921 tggatgcagt gaaaaaggga ttgggaggct ccgcaatccc tgtgaagaga ggcgccaccg
     1981 gcgagacaaa ggtgtttacc ggcaactcta atagccctaa gtccccaaca aagggcggct
     2041 gctctggagg cagcaccaac ctgtctgaca tcatcgagaa ggagacaggc aagcagctgg
     2101 tcatccagga gagcatcctg atgctgcccg aagaagtcga agaagtgatc ggaaacaagc
     2161 ctgagagcga tatcctggtc cataccgcct acgacgagag taccgacgaa aatgtgatgc
     2221 tgctgacatc cgacgcccca gagtataagc cctgggctct ggtcatccag gattccaacg
     2281 gagagaacaa aatcaaaatg ctgtaatcta gagggcccgt ttaaacccgc tgatcagcct
     2341 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2401 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     2461 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     2521 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct cgagggggtt
     2581 ggggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg
     2641 tggttccggg aaacgcagcg gcgccgaccc tgggactcgc acattcttca cgtccgttcg
     2701 cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc
     2761 gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc
     2821 cgcacgtctc actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg
     2881 accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg
     2941 ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc
     3001 gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga
     3061 ccgaatcacc gacctctctc cccaggggga tccaccggag cttaccatga ccgagtacaa
     3121 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
     3181 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
     3241 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     3301 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     3361 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     3421 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     3481 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     3541 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaaa cctccgcgcc
     3601 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     3661 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga
     3721 cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca tcggtacctt taagaccaat
     3781 gacttacaag gcagctgtag atcttagcca ctttctagag tcggggcggc cggccgcttc
     3841 gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     3901 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     3961 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     4021 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc
     4081 cgtcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     4141 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     4201 ccggcagcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4261 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4321 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4381 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4441 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4501 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4561 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4621 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4681 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4741 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4801 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4861 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4921 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4981 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5041 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5101 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5161 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5221 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5281 aatgataccg cgggacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5341 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5401 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5461 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5521 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5581 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5641 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5701 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5761 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5821 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5881 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5941 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6001 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6061 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6121 atttccccga aaagtgccac ctg
//
