LOCUS       sequence-336045-        6839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a truncated VPR-dCjCas9 fusion protein to activate 
            gene expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6839)
  AUTHORS   Zhang X, Lv S, Luo Z, Hu Y, Peng X, Lv J, Zhao S, Feng J, Huang G, 
            Wan QL, Liu J, Huang H, Luan B, Wang D, Zhao X, Lin Y, Zhou Q, Zhang
            ZN, Rong Z
  TITLE     MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional 
            activator, elicits gene expression in vivo.
  JOURNAL   Nucleic Acids Res. 2021 Mar 22. pii: 6179929. doi: 
            10.1093/nar/gkab174.
  PUBMED    33751124
REFERENCE   2  (bases 1 to 6839)
  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: "Nucleic 
            Acids Res. 2021 Mar 22. pii: 6179929. doi: 10.1093/nar/gkab174."
COMMENT     Sequence Label: CMV-VPR-S-L1-dCjCas9 MiniCAFE
FEATURES             Location/Qualifiers
     source          1..6839
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      663..672
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             672..821
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             849..869
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4242..4262
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4263..4289
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             4263..4289
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(4323..4341)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4324..4341)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4361..4378)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4367..4591
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4679..4696)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4850..5438)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4930..4949)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5609..6400,6401..6469))
                     /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     6232..6251
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6470..6574)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6649..6668)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 gggccaccat ggacgcattg gacgattttg atctggatat gctgggaagt gacgccctcg
      721 atgattttga ccttgacatg cttggtagtg atgcccttga tgactttgac ctcgacatgc
      781 tcggcagtga cgcccttgat gatttcgacc tggacatgct gggaggaagc ggaggaggag
      841 gtagcggacc taagaaaaag aggaaggtgg gatccgtgct ggcccagact atggtgccct
      901 ctagtgctat ggtgcctctg gcccagccac ctgctccagc ccctgtgctg accccaggac
      961 caccccagtc actgagcgct ccagtgccca agtctacaca ggccggcgag gggactctga
     1021 gtgaagctct gctgcacctg cagttcgacg ctgatgagga cctgggagct ctgctgggga
     1081 acagcaccga tcccggagtg ttcacagatc tggcctccgt ggacaactct gagtttcagc
     1141 agctgctgaa tcagggcgtg tccatgtctc atagtacagc cgaaccaatg ctgatggagt
     1201 accccgaagc cattacccgg ctggtgaccg gcagccagcg gccccccgac cccgctccaa
     1261 ctcccctggg aaccagcggc ctgcctaatg ggctgtccgg agatgaagac ttctcaagca
     1321 tcgctgatat ggactttagt gccctgctga gtgggtctgg cagcggcagc gacctttccc
     1381 atccgccccc aaggggccat ctggatgagc tgacaaccac acttgagtcc atgaccgagg
     1441 atctgaacct ggactcaccc ctgaccccgg aattgaacga gattctggat accttcctga
     1501 acgacgagtg cctcttgcat gccatgcata tcagcacagg actgtccatc ttcgacacat
     1561 ctctgtttgg atccaacggt ccgggtggat ctggaggtgg aggttctgga ggacgaatca
     1621 tggccagaat cctggccttc gctatcggca tcagcagcat cggctgggcc ttcagcgaga
     1681 acgacgagct gaaggactgc ggcgtgcgga tcttcaccaa ggtggaaaac cccaagaccg
     1741 gcgagagcct ggccctgccc agaaggctgg ccagaagcgc ccggaagaga ctggccagac
     1801 ggaaggcccg gctgaaccac ctgaagcacc tgatcgccaa cgagttcaag ctgaactacg
     1861 aggactacca gagcttcgac gagtccctgg ccaaggccta caagggcagc ctgatcagcc
     1921 cctacgagct gcggttccgg gccctgaacg agctgctgag caagcaggac ttcgccagag
     1981 tgatcctgca cattgccaag cggagaggct acgacgacat caagaacagc gacgacaaag
     2041 agaagggcgc catcctgaag gccatcaagc agaacgagga aaagctggcc aactaccagt
     2101 ccgtgggcga gtacctgtac aaagagtact tccagaagtt caaagagaac agcaaagaat
     2161 tcaccaacgt gcggaacaag aaagaaagct acgagcggtg tatcgcccag agcttcctga
     2221 aggatgagct gaagctgatc ttcaagaagc agagagagtt cggcttcagc ttcagcaaga
     2281 aattcgagga agaggtgctg agcgtcgcct tctacaagag agccctgaag gacttcagcc
     2341 acctcgtggg caactgcagc ttcttcaccg acgagaagag agcccccaag aacagccccc
     2401 tggccttcat gttcgtggcc ctgacccgga tcatcaacct gctgaacaat ctgaagaaca
     2461 ccgagggcat cctgtacacc aaggacgacc tgaacgccct gctgaatgag gtgctgaaga
     2521 acggcaccct gacctacaag cagaccaaga agctgctggg cctgagcgac gactacgagt
     2581 ttaagggcga gaagggcacc tacttcatcg agttcaagaa gtacaaagag ttcatcaagg
     2641 ccctgggcga gcacaacctg agccaggacg atctgaatga gatcgccaag gacatcaccc
     2701 tgatcaagga cgagattaag ctgaagaagg ccctggccaa atacgacctg aatcagaacc
     2761 agatcgacag cctgagcaag ctggaattca aggatcacct gaacatcagc ttcaaggctc
     2821 tgaagctggt cacccccctg atgctggaag gcaagaagta cgacgaggcc tgcaacgagc
     2881 tgaacctgaa ggtggccatc aacgaggaca agaaggactt cctgcccgcc ttcaacgaaa
     2941 cctactacaa ggacgaagtg accaaccccg tggtgctgcg ggccatcaaa gaataccgga
     3001 aggtgctgaa tgccctgctc aagaaatacg gcaaggtgca caagatcaac atcgagctgg
     3061 cccgggaagt gggcaagaac cacagccagc gggccaaggg aagcaagaac ctgcccacca
     3121 agaaacagaa gagaatcctg gacaagaatt acaaggacaa agagcagaag aacttcaagg
     3181 accggaacct gaacgacacc cggtatatcg cccggctggt gctgaactac acaaaggact
     3241 acctggattt cctgcccctg tccgacgacg agaacaccaa gctgaacgat acccagaaag
     3301 gctccaaggt gcacgtggaa gccaagagcg gcatgctgac cagcgccctg agacacacct
     3361 ggggcttcag cgccaaggat cggaacaacc atctgcacca cgccatcgac gccgtgatca
     3421 ttgcctacgc caacaacagc atcgtgaagg ccttctccga cttcaagaaa gaacaggaaa
     3481 gcaacagcgc cgagctgtac gccaagaaga tctctgagct ggactacaag aacaagcgga
     3541 agttcttcga gcccttcagc ggcttccggc agaaggtgct ggataagatc gacgagatct
     3601 tcgtgtccaa gcccgagcgg aagaagccct ctggcgccct gcacgaggaa accttcagaa
     3661 aagaggaaga gttctaccag tcctacggcg gcaaagaagg cgtgctgaag gccctcgagc
     3721 tgggcaagat cagaaaagtg aacggcaaga tcgtgaagaa cggggacatg ttccgggtgg
     3781 acatcttcaa gcacaaaaag accaacaagt tctacgccgt gcccatctac acaatggact
     3841 tcgccctgaa ggtgctgccc aacaaggccg tggcccggtc caagaagggc gagatcaagg
     3901 actggattct gatggacgag aactacgagt tctgctttag cctgtacaag gactccctga
     3961 tcctgatcca gaccaaggac atgcaggaac ccgagttcgt ctactacaac gccttcacca
     4021 gcagcaccgt gtccctgatc gtgtctaagc acgacaacaa gttcgagaca ctgagcaaga
     4081 accagaagat cctgttcaag aacgccaacg agaaagaagt gatcgccaag agcatcggca
     4141 tccagaatct gaaggtgttc gagaagtaca tcgtgtccgc cctgggagaa gtgacaaagg
     4201 ccgagttccg gcagagagag gacttcaaaa agtccggacc tccaaagaaa aagagaaaag
     4261 tataccccta cgacgtgccc gactacgcct aataactcga gcatgcatct agagggccct
     4321 attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt gccttctagt
     4381 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     4441 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     4501 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     4561 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctgcatt
     4621 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4681 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4741 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4801 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4861 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4921 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4981 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5041 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5101 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5161 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5221 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5281 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5341 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5401 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5461 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5521 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5581 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5641 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5701 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5761 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5821 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5881 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5941 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6001 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6061 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6121 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6181 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6241 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6301 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6361 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6421 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6481 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6541 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6601 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     6661 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     6721 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     6781 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgtt
//
