LOCUS       Exported                8502 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor ZNF84.
ACCESSION   .
VERSION     .
KEYWORDS    pZNF84.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8502)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8502)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8502
                     /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"
     gap             572..589
                     /estimated_length=18
     intron          728..955
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      967..976
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             976..1041
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1048..1068
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1093..5193
                     /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             5194..5241
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5269..5286)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5275..5482
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5491..5631
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5491..5620
                     /label=AAV2 ITR
     rep_origin      5706..6161
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5793..5812)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6003..6024
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6178..6197)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6297..6319
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6357..6375)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6443..6547
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6548..7408
                     /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(6766..6785)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7579..8167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8068..8087
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8229..8469
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8229..8249
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8400..8419
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8499..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 gnnnnnnnnn nnnnnnnnng ggcnnggcga
      601 ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg
      661 aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg
      721 gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg
      781 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc
      841 ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg
      901 tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg
      961 accggtgcca ccatggacta taaggaccac gacggagact acaaggatca tgatattgat
     1021 tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga
     1081 gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
     1141 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     1201 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     1261 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     1321 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     1381 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     1441 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     1501 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     1561 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     1621 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     1681 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     1741 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     1801 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     1861 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     1921 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     1981 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     2041 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     2101 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     2161 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     2221 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     2281 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     2341 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     2401 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     2461 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     2521 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     2581 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     2641 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     2701 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     2761 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     2821 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     2881 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     2941 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     3001 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     3061 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     3121 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     3181 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     3241 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     3301 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     3361 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     3421 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     3481 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     3541 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     3601 gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     3661 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     3721 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     3781 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     3841 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     3901 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     3961 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     4021 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     4081 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     4141 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     4201 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     4261 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     4321 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     4381 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     4441 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     4501 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     4561 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     4621 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     4681 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     4741 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     4801 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     4861 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     4921 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     4981 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     5041 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     5101 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     5161 acacggatcg acctgtctca gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc
     5221 ggccaggcaa aaaagaaaaa gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc
     5281 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     5341 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     5401 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     5461 agaatagcag gcatgctggg gagcggccgc aggaacccct agtgatggag ttggccactc
     5521 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     5581 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     5641 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     5701 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     5761 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct
     5821 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     5881 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     5941 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     6001 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga
     6061 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa
     6121 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     6181 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     6241 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     6301 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     6361 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     6421 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     6481 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     6541 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     6601 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     6661 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     6721 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     6781 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     6841 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     6901 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     6961 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     7021 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     7081 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     7141 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     7201 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     7261 tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     7321 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     7381 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     7441 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     7501 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     7561 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     7621 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     7681 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     7741 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     7801 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     7861 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     7921 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     7981 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     8041 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     8101 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     8161 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     8221 tcacatgtga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     8281 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     8341 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     8401 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     8461 ggaaaggacg aaacaccgct actattaact gtattcctgt tt
//
