LOCUS       Exported                8469 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor ERF.
ACCESSION   .
VERSION     .
KEYWORDS    pERF.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8469)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8469)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8469
                     /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          695..922
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      934..943
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             943..1008
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1015..1035
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1060..5160
                     /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             5161..5208
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5236..5253)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5242..5449
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5458..5598
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5458..5587
                     /label=AAV2 ITR
     rep_origin      5673..6128
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5760..5779)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5970..5991
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6145..6164)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6264..6286
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6324..6342)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6410..6514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6515..7375
                     /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(6733..6752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7546..8134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8035..8054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8196..8436
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8196..8216
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8367..8386
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8466..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 ggggggcggg gcgaggcgga gaggtgcggc
      601 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
      661 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc
      721 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
      781 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc tgagcaagag
      841 gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc tggagcacct
      901 gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa ggaccacgac
      961 ggagactaca aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag
     1021 aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta cagcatcggc
     1081 ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
     1141 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
     1201 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
     1261 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     1321 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     1381 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     1441 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     1501 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     1561 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     1621 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     1681 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     1741 ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg
     1801 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     1861 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     1921 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     1981 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
     2041 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     2101 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
     2161 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     2221 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     2281 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
     2341 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     2401 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     2461 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
     2521 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
     2581 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
     2641 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc
     2701 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
     2761 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
     2821 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
     2881 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
     2941 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
     3001 aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac
     3061 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
     3121 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
     3181 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
     3241 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
     3301 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
     3361 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
     3421 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
     3481 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
     3541 gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag
     3601 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac
     3661 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac
     3721 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg
     3781 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
     3841 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag
     3901 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
     3961 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
     4021 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc
     4081 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
     4141 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
     4201 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
     4261 accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc
     4321 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag
     4381 tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct
     4441 aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa
     4501 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc
     4561 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa
     4621 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac
     4681 ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg
     4741 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc
     4801 cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag
     4861 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac
     4921 aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat
     4981 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac
     5041 accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc
     5101 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac
     5161 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagta agaattccta
     5221 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     5281 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     5341 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     5401 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg
     5461 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     5521 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     5581 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5641 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     5701 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     5761 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5821 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5881 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5941 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     6001 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     6061 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     6121 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     6181 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     6241 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     6301 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     6361 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6421 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     6481 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     6541 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     6601 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     6661 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     6721 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     6781 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     6841 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6901 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6961 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     7021 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     7081 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     7141 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     7201 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     7261 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     7321 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     7381 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     7441 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     7501 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     7561 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     7621 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     7681 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     7741 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     7801 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     7861 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     7921 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     7981 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     8041 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     8101 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     8161 gttcctggcc ttttgctggc cttttgctca catgtgaggg cctatttccc atgattcctt
     8221 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     8281 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     8341 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     8401 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtggag caccgagact
     8461 cctgagttt
//
