LOCUS       sequence-343236-        6377 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 6377)
  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 6377)
  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(G1397N)-UGI-Puro
FEATURES             Location/Qualifiers
     source          1..6377
                     /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             2289..2540
                     /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(2572..2589)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2578..2802
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2809..3319
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3238..3256
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3341..3940
                     /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(3341..3360)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3837..3857
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4087..4208
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4098..4117)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4152..4171
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4456..4473)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4627..5215)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4707..4726)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5386..6177,6178..6246))
                     /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     6009..6028
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6247..6351)
                     /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 ccggctccta cgccctgggc ccatatcaga
     1981 tctctgcccc acagctgcca gcatacaacg gacagaccgt gggcacattc tactatgtga
     2041 atgacgccgg cggcctggag tctaaggtgt ttagctccgg cggcccaacc ccctacccta
     2101 actatgccaa tgcaggacac gtggagggac agagcgccct gttcatgcgg gataacggca
     2161 tctccgaggg cctggtgttc cacaacaatc ccgagggcac ctgcggcttt tgcgtgaaca
     2221 tgaccgagac actgctgccc gagaatgcca agatgacagt ggtgccacct gagggatctg
     2281 gaggcagcac caacctgtct gacatcatcg agaaggagac aggcaagcag ctggtcatcc
     2341 aggagagcat cctgatgctg cccgaagaag tcgaagaagt gatcggaaac aagcctgaga
     2401 gcgatatcct ggtccatacc gcctacgacg agagtaccga cgaaaatgtg atgctgctga
     2461 catccgacgc cccagagtat aagccctggg ctctggtcat ccaggattcc aacggagaga
     2521 acaaaatcaa aatgctgtaa tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     2581 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2641 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2701 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2761 aagacaatag caggcatgct ggggatgcgg tgggctctat ggctcgaggg ggttggggtt
     2821 gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc
     2881 cgggaaacgc agcggcgccg accctgggac tcgcacattc ttcacgtccg ttcgcagcgt
     2941 cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct
     3001 aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg
     3061 tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg
     3121 atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg
     3181 ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg
     3241 ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat
     3301 caccgacctc tctccccagg gggatccacc ggagcttacc atgaccgagt acaagcccac
     3361 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     3421 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     3481 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     3541 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     3601 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     3661 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     3721 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     3781 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gaaacctccg cgccccgcaa
     3841 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     3901 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca
     3961 gcgcccgacc gaaaggagcg cacgacccca tgcatcggta cctttaagac caatgactta
     4021 caaggcagct gtagatctta gccactttct agagtcgggg cggccggccg cttcgagcag
     4081 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     4141 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     4201 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     4261 aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatccgtcga
     4321 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     4381 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     4441 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4501 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4561 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4621 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4681 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4741 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4801 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4861 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4921 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4981 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5041 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5101 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5161 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5221 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5281 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5341 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5401 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5461 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5521 accgcgggac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5581 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5641 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5701 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5761 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5821 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5881 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5941 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6001 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6061 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6121 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6181 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6241 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6301 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6361 ccgaaaagtg ccacctg
//
