LOCUS       Exported                7785 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses dCas9-VP64 fusion.
ACCESSION   .
VERSION     .
KEYWORDS    pSL690
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7785)
  AUTHORS   Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK
  TITLE     CRISPR RNA-guided activation of endogenous human genes.
  JOURNAL   Nat Methods. 2013 Jul 25. doi: 10.1038/nmeth.2598.
  PUBMED    23892898
REFERENCE   2  (bases 1 to 7785)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7785
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      403..412
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             409..4512
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4519..4539
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4546..4611
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4642..4791
                     /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             4800..4817
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4840..4857)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4846..5070
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5141..5157)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5141..5157)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5154..5176)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5165..5181
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5189..5219)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5234..5255
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5372..5389)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5543..6131)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5623..5642)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6302..7162)
                     /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     6925..6944
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7163..7267)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7346..7365)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7537..131
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggataagaaa
      421 tactcaatag gcttagctat cggcacaaat agcgtcggat gggcggtgat cactgatgaa
      481 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa
      541 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc
      601 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag
      661 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct
      721 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat
      781 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat
      841 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt
      901 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta
      961 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt
     1021 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat
     1081 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg
     1141 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta
     1201 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat
     1261 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat
     1321 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc
     1381 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca
     1441 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat
     1501 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat
     1561 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc
     1621 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga
     1681 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg
     1741 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg
     1801 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa
     1861 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat
     1921 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg
     1981 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag
     2041 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta
     2101 aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa
     2161 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa
     2221 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc
     2281 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat
     2341 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga
     2401 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa
     2461 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt
     2521 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt
     2581 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt
     2641 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca
     2701 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc
     2761 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact
     2821 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg
     2881 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatgccat tgttccacaa
     2941 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt
     3001 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga
     3061 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa
     3121 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact
     3181 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat
     3241 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct
     3301 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc
     3361 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt
     3421 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag
     3481 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac
     3541 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa
     3601 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc
     3661 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga
     3721 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa
     3781 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta
     3841 gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta
     3901 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct
     3961 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt
     4021 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat
     4081 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag
     4141 ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat
     4201 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat
     4261 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa
     4321 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt
     4381 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat
     4441 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag
     4501 ctaggaggtg acggttctcc caagaagaag aggaaagtct cgagcgacta caaagaccat
     4561 gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa ggctgcagga
     4621 ggcggtggaa gcgggcgcgc cgacgcgctg gacgatttcg atctcgacat gctgggttct
     4681 gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga tgactttgat
     4741 ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt ataaccggtc
     4801 atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     4861 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     4921 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     4981 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     5041 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     5101 cgataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     5161 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     5221 cctagggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     5281 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5341 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5401 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5461 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5521 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5581 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5641 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5701 ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca
     5761 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5821 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5881 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5941 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     6001 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6061 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6121 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6181 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6241 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6301 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6361 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6421 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6481 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6541 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6601 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6661 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6721 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6781 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6841 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6901 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6961 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7021 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7081 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7141 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7201 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7261 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatcgatctc
     7321 ccgatcccct agggtcgact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     7381 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     7441 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     7501 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
     7561 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     7621 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     7681 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     7741 actatttacg gtaaactgcc cacttggcag tacatcaagt gtatc
//
