LOCUS       Exported                7451 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  dCas9VP96 on pmax expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    pAC92-pmax-dCas9VP96
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7451)
  AUTHORS   Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan 
            S, Shivalila CS, Dadon DB, Jaenisch R
  TITLE     Multiplexed activation of endogenous genes by CRISPR-on, an 
            RNA-guided transcriptional activator system.
  JOURNAL   Cell Res. 2013 Aug 27. doi: 10.1038/cr.2013.122.
  PUBMED    23979020
REFERENCE   2  (bases 1 to 7451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7451
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          813..945
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     protein_bind    960..984
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1008..1034
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1038..1058
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1068..5168
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5172..5192
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5226..5453
                     /codon_start=1
                     /product="6 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP96
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDMLGSDALDDFDLDMLGSDALDDFDLDML"
     protein_bind    complement(5481..5505)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5558..5599
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(5625..5644)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    5654..5735
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5679..5698
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(5933..6520)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6013..6032)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6601..7395)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6712..6731)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7322..7341
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg
      721 tcgtttagtg aaccgtcaga tcactagtag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctc gactgatcac aggtaagtat caaggttaca agacaggttt
      841 aaggaggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg
      901 cacctattgg tcttactgac atccactttg cctttctctc cacagggaaa aaacaattga
      961 caagtttgta caaaaaagca ggctccgaat tcaccggtgc caccatgtac ccatacgatg
     1021 ttccagatta cgcttcgccg aagaaaaagc gcaaggtcga agcgtccgac aagaagtaca
     1081 gcatcggcct ggccatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
     1141 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
     1201 acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga
     1261 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
     1321 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
     1381 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
     1441 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
     1501 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
     1561 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     1621 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     1681 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     1741 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc
     1801 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     1861 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     1921 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     1981 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     2041 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     2101 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg
     2161 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     2221 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     2281 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
     2341 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
     2401 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
     2461 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
     2521 cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
     2581 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca
     2641 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
     2701 aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
     2761 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
     2821 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
     2881 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
     2941 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
     3001 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
     3061 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
     3121 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
     3181 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
     3241 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
     3301 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
     3361 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
     3421 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
     3481 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
     3541 aggaactgga catcaaccgg ctgtccgact acgatgtgga cgccatcgtg cctcagagct
     3601 ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca
     3661 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc
     3721 tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag
     3781 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
     3841 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
     3901 atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
     3961 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
     4021 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
     4081 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
     4141 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
     4201 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
     4261 acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag
     4321 tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
     4381 gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact
     4441 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
     4501 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
     4561 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
     4621 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
     4681 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
     4741 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
     4801 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc
     4861 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
     4921 atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg
     4981 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
     5041 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
     5101 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
     5161 gaggcgacag ccccaagaag aagagaaagg tggaggccag cgggccggcc ggatccgggc
     5221 gcgccgacgc gctggacgat ttcgatctcg acatgctggg ttctgatgcc ctcgatgact
     5281 ttgacctgga tatgttggga agcgacgcat tggatgactt tgatctggac atgctcggct
     5341 ccgatgctct ggacgatttc gatctcgata tgttagggtc agacgcactg gatgatttcg
     5401 accttgatat gttgggaagc gatgcccttg atgatttcga tttggatatg ttgtatatcg
     5461 attgattaat taagaattcg acccagcttt cttgtacaaa gtggttgata tccagcacag
     5521 tggcggccgc tcgagtctag agggcccgcg gttcgaaggt aagcctatcc ctaaccctct
     5581 cctcggtctc gattctacgc gtaccggtta gcaattgttt tttcgatgag tttggacaaa
     5641 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     5701 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     5761 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat
     5821 gtggtactta agagggggag accaaagggc gagacgttaa ggcctcacgt gacatgtgag
     5881 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5941 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6001 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6061 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6121 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6181 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6241 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6301 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6361 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6421 aaagagttgg tagctcttga tccggcaaac aaaccacgct ggtagcggtg gtttttttgt
     6481 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6541 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgccgtc
     6601 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
     6661 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
     6721 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
     6781 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
     6841 gacgagatcc tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc
     6901 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
     6961 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
     7021 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
     7081 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
     7141 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
     7201 cgctgcctcg tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
     7261 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
     7321 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
     7381 atcttgttca atcataatat tattgaagca tttatcaggg ttcgtctcgt cccggtctcc
     7441 tcccatgcat g
//
