LOCUS       sequence-336055-        8224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dpy-30 promoter-driven expression of a truncated VPR-dCjCas9 fusion 
            protein (MiniCAFE) for activation of gene expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8224)
  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 8224)
  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: Pdpy-30-miniCAFE
FEATURES             Location/Qualifiers
     source          1..8224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      2028..2037
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2037..2186
                     /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             2214..2234
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5607..5627
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5628..5654
                     /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             5628..5654
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(5688..5706)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5689..5706)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5726..5743)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5732..5956
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6044..6061)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6215..6803)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6295..6314)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6974..7765,7766..7834))
                     /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     7597..7616
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7835..7939)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8014..8033)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agtttatgtt gtttttttaa gacatatacc gaacaatttt aaatgtaagg tatcgacagt
       61 ttgatcaaat tttggtaagt gtcagtttga ttttttgaat aaacttctaa acgaaactgc
      121 ctttctcaag cgaaacttta tcaagcaaat gaccaaaagg agagtaatta gacagtttta
      181 acggttcaat attaaaacgt aaaaattttc aaaaaaaagt gagatgagga tgagggaaaa
      241 gttgaaaagt tcctatccgt tagtaattcg atactttaac ttttctacta aacgaaaaaa
      301 tgagaagatg tttggtgtat tctgctttca aaaattgtgt tgtaagcttt cgggtaaagt
      361 aaaagtaaag tgggttatgt ataattcatc tctaaaaaag aggttaaatt tacaatgaac
      421 accagaattt ttatattgat tgtgcaccgt tcggattctg aaaattaaag aaattgaccg
      481 aatattttta gagaaaatat gattcagttg tagttgtttt gtcaaatagt caagtaaatg
      541 atacaaatcc agaaaactcc aattaccaac gaacaagata ttccaacaaa aattgtttta
      601 tttacagtaa aaaagataag gaaaaagagt tatctaaatc ttaaggtaca ttgaccaaaa
      661 ttttatgttc taattatgtt ttcattgttt taacttaaac ttaatttaaa ctgttttaat
      721 ttaattgtag ggattcgaat attcgagaca gctccaaatt tcttgacaat cccaattttt
      781 caaaagtttg ccaatttacc aaaacttcac aaacaaattc aaaagtgtct tatctagatt
      841 tttaaaaatt tgttcttctc cacttgttct cttaataaaa taaattctaa attttgaaca
      901 ctaattagtg cactaatcaa tgttttgaat tttgaataaa ataattttaa aattttaaat
      961 gttgtaaatt tttagtgata ctgtaggtac aaggaaacta ttttattgaa ttcttgatca
     1021 aagaaggtag gcagtttata taaataatct tattgttaga taccattttg aatatatgtt
     1081 tcagttccag aaaaataagc tatacatagt atcgatttgg aacatatata acgaaaacag
     1141 acgtcatgaa atcacacatt ggaaaagtga gaaagttgtt gagtagagga ggtgacaaaa
     1201 gtgagatgcc aactcactta tccattctac tcgttttgtg ctcttttcca ctttctgatg
     1261 tctgctcatc ttcttattct tcaagcgttt tgtctattct cacacctctc caatctttca
     1321 gtttctgttt ttaatgtcta gacacgcgtg taacgtacaa cataaatgtt tgacgtcaac
     1381 cagatgtagt acaataaaaa atatattcag attacctaga aaattacttc ggtcatgacc
     1441 accaattttt tgatttttcg attttcgaaa ccgacatcgt tgaagaacat tcaggtcata
     1501 attttttatt tgaaaaataa tgttttctat gtattaattc ggtttagtcg atcgaatatg
     1561 tacagttgag aattactaga gtacggtgcg caagtatgca aacttgcgta acggtgttta
     1621 cacatttgtc acgtttctct gcgtctcttc gtctcaacaa tgcttgcact ccgtcacatt
     1681 ttcgaattgt cggccgatat cggtatttag ataaattcag tgctctcctt tttttctcca
     1741 ttttactttc atcaatcttt tttaaaaatc aatattcagc aaactattga aaaattaatt
     1801 caaaaaacac gactggtcgg aagttatttg cataatcaat agaaatttcg ttttccgctc
     1861 gctgtcagtg ttttgtcctg tttgtatttc ccagtgcact tttcgagtgg ctcaagtttt
     1921 tatctatttt cctgcaaact tgcatatatt gttttcttcc agaagccttt gatatctcaa
     1981 taattttata cttgtttcag aaatcgagca aaaaccaaga agcttgggcc accatggatg
     2041 cattggacga ttttgatctg gatatgctgg gaagtgacgc cctcgatgat tttgaccttg
     2101 acatgcttgg tagtgatgcc cttgatgact ttgacctcga catgctcggc agtgacgccc
     2161 ttgatgattt cgacctggac atgctgggag gaagcggagg aggaggtagc ggacctaaga
     2221 aaaagaggaa ggtgggatcc gtgctggccc agactatggt gccctctagt gctatggtgc
     2281 ctctggccca gccacctgct ccagcccctg tgctgacccc aggaccaccc cagtcactga
     2341 gcgctccagt gcccaagtct acacaggccg gcgaggggac tctgagtgaa gctctgctgc
     2401 acctgcagtt cgacgctgat gaggacctgg gagctctgct ggggaacagc accgatcccg
     2461 gagtgttcac agatctggcc tccgtggaca actctgagtt tcagcagctg ctgaatcagg
     2521 gcgtgtccat gtctcatagt acagccgaac caatgctgat ggagtacccc gaagccatta
     2581 cccggctggt gaccggcagc cagcggcccc ccgaccccgc tccaactccc ctgggaacca
     2641 gcggcctgcc taatgggctg tccggagatg aagacttctc aagcatcgct gatatggact
     2701 ttagtgccct gctgagtggg tctggcagcg gcagcgacct ttcccatccg cccccaaggg
     2761 gccatctgga tgagctgaca accacacttg agtccatgac cgaggatctg aacctggact
     2821 cacccctgac cccggaattg aacgagattc tggatacctt cctgaacgac gagtgcctct
     2881 tgcatgccat gcatatcagc acaggactgt ccatcttcga cacatctctg tttggatcca
     2941 acggtccggg tggatctgga ggtggaggtt ctggaggacg aatcatggcc agaatcctgg
     3001 ccttcgctat cggcatcagc agcatcggct gggccttcag cgagaacgac gagctgaagg
     3061 actgcggcgt gcggatcttc accaaggtgg aaaaccccaa gaccggcgag agcctggccc
     3121 tgcccagaag gctggccaga agcgcccgga agagactggc cagacggaag gcccggctga
     3181 accacctgaa gcacctgatc gccaacgagt tcaagctgaa ctacgaggac taccagagct
     3241 tcgacgagtc cctggccaag gcctacaagg gcagcctgat cagcccctac gagctgcggt
     3301 tccgggccct gaacgagctg ctgagcaagc aggacttcgc cagagtgatc ctgcacattg
     3361 ccaagcggag aggctacgac gacatcaaga acagcgacga caaagagaag ggcgccatcc
     3421 tgaaggccat caagcagaac gaggaaaagc tggccaacta ccagtccgtg ggcgagtacc
     3481 tgtacaaaga gtacttccag aagttcaaag agaacagcaa agaattcacc aacgtgcgga
     3541 acaagaaaga aagctacgag cggtgtatcg cccagagctt cctgaaggat gagctgaagc
     3601 tgatcttcaa gaagcagaga gagttcggct tcagcttcag caagaaattc gaggaagagg
     3661 tgctgagcgt cgccttctac aagagagccc tgaaggactt cagccacctc gtgggcaact
     3721 gcagcttctt caccgacgag aagagagccc ccaagaacag ccccctggcc ttcatgttcg
     3781 tggccctgac ccggatcatc aacctgctga acaatctgaa gaacaccgag ggcatcctgt
     3841 acaccaagga cgacctgaac gccctgctga atgaggtgct gaagaacggc accctgacct
     3901 acaagcagac caagaagctg ctgggcctga gcgacgacta cgagtttaag ggcgagaagg
     3961 gcacctactt catcgagttc aagaagtaca aagagttcat caaggccctg ggcgagcaca
     4021 acctgagcca ggacgatctg aatgagatcg ccaaggacat caccctgatc aaggacgaga
     4081 ttaagctgaa gaaggccctg gccaaatacg acctgaatca gaaccagatc gacagcctga
     4141 gcaagctgga attcaaggat cacctgaaca tcagcttcaa ggctctgaag ctggtcaccc
     4201 ccctgatgct ggaaggcaag aagtacgacg aggcctgcaa cgagctgaac ctgaaggtgg
     4261 ccatcaacga ggacaagaag gacttcctgc ccgccttcaa cgaaacctac tacaaggacg
     4321 aagtgaccaa ccccgtggtg ctgcgggcca tcaaagaata ccggaaggtg ctgaatgccc
     4381 tgctcaagaa atacggcaag gtgcacaaga tcaacatcga gctggcccgg gaagtgggca
     4441 agaaccacag ccagcgggcc aagggaagca agaacctgcc caccaagaaa cagaagagaa
     4501 tcctggacaa gaattacaag gacaaagagc agaagaactt caaggaccgg aacctgaacg
     4561 acacccggta tatcgcccgg ctggtgctga actacacaaa ggactacctg gatttcctgc
     4621 ccctgtccga cgacgagaac accaagctga acgataccca gaaaggctcc aaggtgcacg
     4681 tggaagccaa gagcggcatg ctgaccagcg ccctgagaca cacctggggc ttcagcgcca
     4741 aggatcggaa caaccatctg caccacgcca tcgacgccgt gatcattgcc tacgccaaca
     4801 acagcatcgt gaaggccttc tccgacttca agaaagaaca ggaaagcaac agcgccgagc
     4861 tgtacgccaa gaagatctct gagctggact acaagaacaa gcggaagttc ttcgagccct
     4921 tcagcggctt ccggcagaag gtgctggata agatcgacga gatcttcgtg tccaagcccg
     4981 agcggaagaa gccctctggc gccctgcacg aggaaacctt cagaaaagag gaagagttct
     5041 accagtccta cggcggcaaa gaaggcgtgc tgaaggccct cgagctgggc aagatcagaa
     5101 aagtgaacgg caagatcgtg aagaacgggg acatgttccg ggtggacatc ttcaagcaca
     5161 aaaagaccaa caagttctac gccgtgccca tctacacaat ggacttcgcc ctgaaggtgc
     5221 tgcccaacaa ggccgtggcc cggtccaaga agggcgagat caaggactgg attctgatgg
     5281 acgagaacta cgagttctgc tttagcctgt acaaggactc cctgatcctg atccagacca
     5341 aggacatgca ggaacccgag ttcgtctact acaacgcctt caccagcagc accgtgtccc
     5401 tgatcgtgtc taagcacgac aacaagttcg agacactgag caagaaccag aagatcctgt
     5461 tcaagaacgc caacgagaaa gaagtgatcg ccaagagcat cggcatccag aatctgaagg
     5521 tgttcgagaa gtacatcgtg tccgccctgg gagaagtgac aaaggccgag ttccggcaga
     5581 gagaggactt caaaaagtcc ggacctccaa agaaaaagag aaaagtatac ccctacgacg
     5641 tgcccgacta cgcctaataa ctcgagcatg catctagagg gccctattct atagtgtcac
     5701 ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5761 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5821 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5881 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     5941 gcggtgggct ctatggcttc tgaggcggaa agaaccagct gcattaatga atcggccaac
     6001 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     6061 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6121 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6181 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6241 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6301 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6361 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6421 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6481 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6541 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6601 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6661 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6721 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6781 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6841 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6901 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6961 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     7021 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     7081 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     7141 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7201 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7261 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7321 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7381 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7441 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7501 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7561 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7621 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7681 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7741 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7801 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7861 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7921 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     7981 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     8041 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     8101 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     8161 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     8221 tagt
//
