LOCUS       Exported                8460 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor TEAD1.
ACCESSION   .
VERSION     .
KEYWORDS    pTEAD1.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8460)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8460)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8460
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        169..454
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          686..913
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      925..934
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             934..999
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1006..1026
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1051..5151
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5152..5199
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5227..5244)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5233..5440
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5449..5589
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5449..5578
                     /label=AAV2 ITR
     rep_origin      5664..6119
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5751..5770)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5961..5982
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6136..6155)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6255..6277
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6315..6333)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6401..6505
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6506..7366
                     /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     complement(6724..6743)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7537..8125
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8026..8045
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8187..8427
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8187..8207
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8358..8377
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8457..72
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
       61 ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
      121 tttttagcgc gtgcgccaat tctgcagaca aatggctcta gaggtacccg ttacataact
      181 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt
      241 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      301 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      361 taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca
      421 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
      481 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
      541 attttgtgca gcgatggggg cggggggggg ggcgaggcgg agaggtgcgg cggcagccaa
      601 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
      661 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
      721 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
      781 gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt
      841 taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat
      901 cacttttttt caggttggac cggtgccacc atggactata aggaccacga cggagactac
      961 aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg
     1021 aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc
     1081 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     1141 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
     1201 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     1261 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     1321 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     1381 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     1441 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     1501 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     1561 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     1621 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     1681 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     1741 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac
     1801 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     1861 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     1921 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     1981 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     2041 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     2101 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     2161 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     2221 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     2281 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     2341 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     2401 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     2461 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     2521 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     2581 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     2641 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     2701 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     2761 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     2821 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     2881 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     2941 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     3001 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     3061 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     3121 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     3181 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     3241 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     3301 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     3361 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     3421 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     3481 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     3541 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc
     3601 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     3661 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     3721 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     3781 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     3841 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     3901 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     3961 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
     4021 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
     4081 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
     4141 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     4201 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     4261 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     4321 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     4381 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     4441 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     4501 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     4561 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     4621 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     4681 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     4741 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     4801 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     4861 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     4921 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     4981 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     5041 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     5101 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg
     5161 gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcct agagctcgct
     5221 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     5281 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     5341 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     5401 agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag
     5461 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     5521 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     5581 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     5641 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     5701 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     5761 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5821 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     5881 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5941 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     6001 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     6061 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     6121 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     6181 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     6241 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     6301 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     6361 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     6421 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     6481 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     6541 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     6601 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     6661 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     6721 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     6781 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     6841 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6901 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6961 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     7021 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     7081 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     7141 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     7201 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     7261 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     7321 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     7381 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     7441 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     7501 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     7561 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     7621 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     7681 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     7741 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7801 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7861 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7921 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7981 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     8041 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     8101 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     8161 cttttgctgg ccttttgctc acatgtgagg gcctatttcc catgattcct tcatatttgc
     8221 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     8281 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     8341 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     8401 tggctttata tatcttgtgg aaaggacgaa acaccgggct tgtaaaggac tgaacagttt
//
