LOCUS       sequence-336053-        7355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of VPR-WT CjCas9 fusion protein to induce orthogonal 
            genes knock out and activation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7355)
  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 7355)
  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: pRGEN-CMV-VPR-WT CjCas9 V-WT
FEATURES             Location/Qualifiers
     source          1..7355
                     /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             885..1427
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             1665..1685
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1692..1712
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1797..4748
                     /codon_start=1
                     /product="Cas9 endonuclease from the Campylobacter jejuni 
                     Type II CRISPR/Cas system (Kim et al., 2016)"
                     /label=CjCas9
                     /translation="MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLA
                     LPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYEL
                     RFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVG
                     EYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKF
                     EEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNT
                     EGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIK
                     ALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFK
                     ALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKE
                     YRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGL
                     KINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVL
                     VFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDR
                     NLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTW
                     GFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKR
                     KFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKAL
                     ELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGE
                     IKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFET
                     LSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK"
     CDS             4758..4778
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4779..4805
                     /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             4779..4805
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(4839..4857)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4840..4857)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4877..4894)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4883..5107
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5195..5212)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5366..5954)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5446..5465)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6125..6916,6917..6985))
                     /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     6748..6767
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6986..7090)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7165..7184)
                     /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 ggatgcttta gacgattttg acttagatat gcttggttca gacgcgttag
      721 acgacttcga cctagacatg ttaggctcag atgcattgga cgacttcgat ttagatatgt
      781 tgggctccga tgccctagat gactttgatc tagatatgct aggaggaagc ggaggaggag
      841 gtagcggacc taagaaaaag aggaaggtgg cggccgctgg atccccttca gggcagatca
      901 gcaaccaggc cctggctctg gcccctagct ccgctccagt gctggcccag actatggtgc
      961 cctctagtgc tatggtgcct ctggcccagc cacctgctcc agcccctgtg ctgaccccag
     1021 gaccacccca gtcactgagc gctccagtgc ccaagtctac acaggccggc gaggggactc
     1081 tgagtgaagc tctgctgcac ctgcagttcg acgctgatga ggacctggga gctctgctgg
     1141 ggaacagcac cgatcccgga gtgttcacag atctggcctc cgtggacaac tctgagtttc
     1201 agcagctgct gaatcagggc gtgtccatgt ctcatagtac agccgaacca atgctgatgg
     1261 agtaccccga agccattacc cggctggtga ccggcagcca gcggcccccc gaccccgctc
     1321 caactcccct gggaaccagc ggcctgccta atgggctgtc cggagatgaa gacttctcaa
     1381 gcatcgctga tatggacttt agtgccctgc tgtcacagat ttcctctagt gggtctggca
     1441 gcggcagcga cctttcccat ccgcccccaa ggggccatct ggatgagctg acaaccacac
     1501 ttgagtccat gaccgaggat ctgaacctgg actcacccct gaccccggaa ttgaacgaga
     1561 ttctggatac cttcctgaac gacgagtgcc tcttgcatgc catgcatatc agcacaggac
     1621 tgtccatctt cgacacatct ctgtttagcc tgggcagcgg ctcccccaag aaaaaacgca
     1681 aggtggaaga tcctaagaaa aagcggaaag tggacggcat tggtagtggg agcaacggca
     1741 gcagcggatc caacggtccg ggtggatctg gaggtggagg ttctggagga cgaaccatgg
     1801 ccagaatcct ggccttcgac atcggcatca gcagcatcgg ctgggccttc agcgagaacg
     1861 acgagctgaa ggactgcggc gtgcggatct tcaccaaggt ggaaaacccc aagaccggcg
     1921 agagcctggc cctgcccaga aggctggcca gaagcgcccg gaagagactg gccagacgga
     1981 aggcccggct gaaccacctg aagcacctga tcgccaacga gttcaagctg aactacgagg
     2041 actaccagag cttcgacgag tccctggcca aggcctacaa gggcagcctg atcagcccct
     2101 acgagctgcg gttccgggcc ctgaacgagc tgctgagcaa gcaggacttc gccagagtga
     2161 tcctgcacat tgccaagcgg agaggctacg acgacatcaa gaacagcgac gacaaagaga
     2221 agggcgccat cctgaaggcc atcaagcaga acgaggaaaa gctggccaac taccagtccg
     2281 tgggcgagta cctgtacaaa gagtacttcc agaagttcaa agagaacagc aaagaattca
     2341 ccaacgtgcg gaacaagaaa gaaagctacg agcggtgtat cgcccagagc ttcctgaagg
     2401 atgagctgaa gctgatcttc aagaagcaga gagagttcgg cttcagcttc agcaagaaat
     2461 tcgaggaaga ggtgctgagc gtcgccttct acaagagagc cctgaaggac ttcagccacc
     2521 tcgtgggcaa ctgcagcttc ttcaccgacg agaagagagc ccccaagaac agccccctgg
     2581 ccttcatgtt cgtggccctg acccggatca tcaacctgct gaacaatctg aagaacaccg
     2641 agggcatcct gtacaccaag gacgacctga acgccctgct gaatgaggtg ctgaagaacg
     2701 gcaccctgac ctacaagcag accaagaagc tgctgggcct gagcgacgac tacgagttta
     2761 agggcgagaa gggcacctac ttcatcgagt tcaagaagta caaagagttc atcaaggccc
     2821 tgggcgagca caacctgagc caggacgatc tgaatgagat cgccaaggac atcaccctga
     2881 tcaaggacga gattaagctg aagaaggccc tggccaaata cgacctgaat cagaaccaga
     2941 tcgacagcct gagcaagctg gaattcaagg atcacctgaa catcagcttc aaggctctga
     3001 agctggtcac ccccctgatg ctggaaggca agaagtacga cgaggcctgc aacgagctga
     3061 acctgaaggt ggccatcaac gaggacaaga aggacttcct gcccgccttc aacgaaacct
     3121 actacaagga cgaagtgacc aaccccgtgg tgctgcgggc catcaaagaa taccggaagg
     3181 tgctgaatgc cctgctcaag aaatacggca aggtgcacaa gatcaacatc gagctggccc
     3241 gggaagtggg caagaaccac agccagcggg ccaagatcga gaaagagcag aacgaaaact
     3301 acaaggccaa gaaggacgct gagctggaat gcgagaagct gggactgaag atcaacagca
     3361 agaacatcct gaagctgcgg ctgttcaaag aacagaaaga gttctgcgcc tacagcggcg
     3421 agaagatcaa gatcagcgat ctgcaggacg agaagatgct ggaaatcgac cacatctacc
     3481 cctacagccg gtccttcgac gacagctaca tgaacaaggt gctggtgttc accaaacaga
     3541 accaggaaaa actgaaccag acccccttcg aggccttcgg caacgacagc gccaagtggc
     3601 agaaaatcga ggtgctggcc aagaacctgc ccaccaagaa acagaagaga atcctggaca
     3661 agaattacaa ggacaaagag cagaagaact tcaaggaccg gaacctgaac gacacccggt
     3721 atatcgcccg gctggtgctg aactacacaa aggactacct ggatttcctg cccctgtccg
     3781 acgacgagaa caccaagctg aacgataccc agaaaggctc caaggtgcac gtggaagcca
     3841 agagcggcat gctgaccagc gccctgagac acacctgggg cttcagcgcc aaggatcgga
     3901 acaaccatct gcaccacgcc atcgacgccg tgatcattgc ctacgccaac aacagcatcg
     3961 tgaaggcctt ctccgacttc aagaaagaac aggaaagcaa cagcgccgag ctgtacgcca
     4021 agaagatctc tgagctggac tacaagaaca agcggaagtt cttcgagccc ttcagcggct
     4081 tccggcagaa ggtgctggat aagatcgacg agatcttcgt gtccaagccc gagcggaaga
     4141 agccctctgg cgccctgcac gaggaaacct tcagaaaaga ggaagagttc taccagtcct
     4201 acggcggcaa agaaggcgtg ctgaaggccc tcgagctggg caagatcaga aaagtgaacg
     4261 gcaagatcgt gaagaacggg gacatgttcc gggtggacat cttcaagcac aaaaagacca
     4321 acaagttcta cgccgtgccc atctacacca tggacttcgc cctgaaggtg ctgcccaaca
     4381 aggccgtggc ccggtccaag aagggcgaga tcaaggactg gattctgatg gacgagaact
     4441 acgagttctg ctttagcctg tacaaggact ccctgatcct gatccagacc aaggacatgc
     4501 aggaacccga gttcgtctac tacaacgcct tcaccagcag caccgtgtcc ctgatcgtgt
     4561 ctaagcacga caacaagttc gagacactga gcaagaacca gaagatcctg ttcaagaacg
     4621 ccaacgagaa agaagtgatc gccaagagca tcggcatcca gaatctgaag gtgttcgaga
     4681 agtacatcgt gtccgccctg ggagaagtga caaaggccga gttccggcag agagaggact
     4741 tcaaaaagtc cggacctcca aagaaaaaga gaaaagtata cccctacgac gtgcccgact
     4801 acgcctaata actcgagcat gcatctagag ggccctattc tatagtgtca cctaaatgct
     4861 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     4921 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4981 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     5041 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     5101 tctatggctt ctgaggcgga aagaaccagc tgcattaatg aatcggccaa cgcgcgggga
     5161 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5221 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5281 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5341 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5401 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5461 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5521 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5581 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5641 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5701 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5761 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5821 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5881 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5941 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6001 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6061 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6121 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6181 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6241 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6301 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6361 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6421 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6481 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6541 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6601 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6661 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6721 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6781 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6841 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6901 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6961 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7021 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7081 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     7141 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     7201 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     7261 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     7321 gctgcttcgc gatgtacggg ccagatatac gcgtt
//
