LOCUS       Exported                8479 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor ZNF586.
ACCESSION   .
VERSION     .
KEYWORDS    pZNF586.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8479)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8479)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8479
                     /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          705..932
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      944..953
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             953..1018
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1025..1045
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1070..5170
                     /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             5171..5218
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5246..5263)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5252..5459
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5468..5608
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5468..5597
                     /label=AAV2 ITR
     rep_origin      5683..6138
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5770..5789)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5980..6001
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6155..6174)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6274..6296
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6334..6352)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6420..6524
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6525..7385
                     /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(6743..6762)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7556..8144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8045..8064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8206..8446
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8206..8226
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8377..8396
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8476..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 ggggcgaggg gcggggcggg gcgaggcgga
      601 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      661 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
      721 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
      781 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
      841 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
      901 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa
      961 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagat
     1021 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta
     1081 cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
     1141 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
     1201 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
     1261 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
     1321 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
     1381 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
     1441 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
     1501 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
     1561 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
     1621 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     1681 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     1741 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag
     1801 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     1861 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     1921 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     1981 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     2041 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     2101 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     2161 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     2221 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     2281 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     2341 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     2401 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     2461 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     2521 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     2581 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     2641 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     2701 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     2761 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     2821 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     2881 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     2941 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     3001 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     3061 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     3121 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     3181 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     3241 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     3301 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     3361 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     3421 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     3481 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     3541 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     3601 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     3661 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     3721 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     3781 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     3841 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     3901 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     3961 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     4021 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     4081 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     4141 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     4201 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     4261 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     4321 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     4381 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     4441 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     4501 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     4561 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     4621 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     4681 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     4741 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     4801 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     4861 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     4921 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     4981 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     5041 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     5101 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     5161 gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagta
     5221 agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5281 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5341 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5401 ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag
     5461 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     5521 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     5581 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     5641 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     5701 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     5761 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5821 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5881 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5941 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     6001 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     6061 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     6121 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6181 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     6241 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     6301 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     6361 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     6421 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6481 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     6541 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6601 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6661 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6721 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6781 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     6841 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6901 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6961 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     7021 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     7081 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     7141 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     7201 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag
     7261 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     7321 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     7381 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     7441 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     7501 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     7561 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7621 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7681 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     7741 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7801 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     7861 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7921 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7981 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     8041 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     8101 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     8161 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtgaggg cctatttccc
     8221 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     8281 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     8341 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     8401 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtctca
     8461 agtgtttaat aagacgttt
//
