LOCUS       sequence-355547-        6302 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 6302)
  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 6302)
  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(G1333C)-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..6302
                     /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             710..733
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1492..1797
                     /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     1676..1695
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2214..2465
                     /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(2497..2514)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2503..2727
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2734..3244
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3163..3181
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3266..3865
                     /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(3266..3285)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3762..3782
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4012..4133
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4023..4042)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4077..4096
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4381..4398)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4552..5140)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4632..4651)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5311..6102,6103..6171))
                     /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     5934..5953
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6172..6276)
                     /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 gcggccgctg actacaagga
      721 tgacgacgat aaaggtagcg tggatctacg cacgctcggc tacagccagc agcaacagga
      781 gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac cacgaggcac tggtcggcca
      841 tgggtttaca cacgcgcaca tcgttgcgct cagccaacac ccggcagcgt tagggaccgt
      901 cgctgtcaag tatcaggaca tgatcgcagc gttgccagag gcgacacacg aagcgatcgt
      961 tggcgtcggc aaacagtggt ccggcgcacg cgctctggag gccttgctca cggtggcggg
     1021 agagttgaga ggtccaccgt tacagttgga cacaggccaa cttctcaaga ttgcaaaacg
     1081 tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc aatgcactga cgggtgcccc
     1141 cctgaacctg accccagaga ccggcttact aaaagccaga taacagtatg cgtatttgcg
     1201 cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag
     1261 aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc
     1321 aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc
     1381 gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc
     1441 ggtttattga aatgaacggc tcttttgctg acgagaacag gggctggtga aatgcagttt
     1501 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
     1561 attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
     1621 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
     1681 atgaccaccg atatggccag tgtgccggtg tccgttatcg gggaagaagt ggctgatctc
     1741 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg
     1801 tcaggctccc ttatacacag ccagggtctc tcgcgttgac caacgaccac ctcgtcgcct
     1861 tggcctgcct cggcggacgt cctgcgctgg atgcagtgaa aaagggattg ggaggctccc
     1921 caacccccta ccctaactat gccaatgcag gacacgtgga gggacagagc gccctgttca
     1981 tgcgggataa cggcatctcc gagggcctgg tgttccacaa caatcccgag ggcacctgcg
     2041 gcttttgcgt gaacatgacc gagacactgc tgcccgagaa tgccaagatg acagtggtgc
     2101 cacctgaggg agcaatccct gtgaagagag gcgccaccgg cgagacaaag gtgtttaccg
     2161 gcaactctaa tagccctaag tccccaacaa agggcggctg ctctggaggc agcaccaacc
     2221 tgtctgacat catcgagaag gagacaggca agcagctggt catccaggag agcatcctga
     2281 tgctgcccga agaagtcgaa gaagtgatcg gaaacaagcc tgagagcgat atcctggtcc
     2341 ataccgccta cgacgagagt accgacgaaa atgtgatgct gctgacatcc gacgccccag
     2401 agtataagcc ctgggctctg gtcatccagg attccaacgg agagaacaaa atcaaaatgc
     2461 tgtaatctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2521 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2581 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2641 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2701 atgctgggga tgcggtgggc tctatggctc gagggggttg gggttgcgcc ttttccaagg
     2761 cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg
     2821 cgccgaccct gggactcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc
     2881 cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc
     2941 cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct
     3001 cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa
     3061 tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc
     3121 ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa
     3181 gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc
     3241 ccagggggat ccaccggagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac
     3301 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     3361 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     3421 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     3481 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     3541 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     3601 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     3661 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     3721 gcgcgccggg gtgcccgcct tcctggaaac ctccgcgccc cgcaacctcc ccttctacga
     3781 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     3841 catgacccgc aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag
     3901 gagcgcacga ccccatgcat cggtaccttt aagaccaatg acttacaagg cagctgtaga
     3961 tcttagccac tttctagagt cggggcggcc ggccgcttcg agcagacatg ataagataca
     4021 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     4081 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     4141 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     4201 agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc gtcgaccgat gcccttgaga
     4261 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     4321 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct cttccgcttc
     4381 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4441 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4501 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4561 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4621 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4681 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4741 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4801 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4861 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4921 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4981 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5041 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5101 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5161 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5221 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5281 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5341 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5401 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gggacccacg
     5461 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5521 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5581 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5641 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5701 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5761 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5821 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5881 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5941 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6001 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6061 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6121 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6181 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6241 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6301 tg
//
