LOCUS       Exported                8480 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor ZNF174.
ACCESSION   .
VERSION     .
KEYWORDS    pZNF174.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8480)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8480)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8480
                     /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          706..933
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      945..954
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             954..1019
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1026..1046
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1071..5171
                     /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             5172..5219
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5247..5264)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5253..5460
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5469..5609
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5469..5598
                     /label=AAV2 ITR
     rep_origin      5684..6139
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5771..5790)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5981..6002
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6156..6175)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6275..6297
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6335..6353)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6421..6525
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6526..7386
                     /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(6744..6763)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7557..8145
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8046..8065
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8207..8447
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8207..8227
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8378..8397
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8477..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 gggggcgagg ggcggggcgg ggcgaggcgg
      601 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
      661 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
      721 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
      781 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      841 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
      901 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata
      961 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
     1021 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt
     1081 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
     1141 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
     1201 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
     1261 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
     1321 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
     1381 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
     1441 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
     1501 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
     1561 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
     1621 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
     1681 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
     1741 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga
     1801 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
     1861 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
     1921 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
     1981 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
     2041 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
     2101 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
     2161 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
     2221 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
     2281 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
     2341 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
     2401 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
     2461 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
     2521 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
     2581 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
     2641 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
     2701 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
     2761 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
     2821 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
     2881 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
     2941 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
     3001 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
     3061 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
     3121 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
     3181 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
     3241 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
     3301 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
     3361 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
     3421 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
     3481 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
     3541 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga
     3601 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
     3661 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
     3721 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
     3781 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
     3841 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
     3901 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
     3961 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
     4021 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
     4081 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
     4141 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     4201 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
     4261 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     4321 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     4381 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     4441 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
     4501 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     4561 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     4621 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     4681 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
     4741 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     4801 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     4861 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     4921 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     4981 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     5041 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
     5101 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     5161 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt
     5221 aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     5281 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     5341 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     5401 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga
     5461 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     5521 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     5581 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     5641 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     5701 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5761 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5821 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5881 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5941 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     6001 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     6061 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     6121 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     6181 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     6241 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     6301 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     6361 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     6421 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     6481 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     6541 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     6601 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     6661 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     6721 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     6781 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6841 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6901 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6961 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     7021 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     7081 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     7141 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     7201 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     7261 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     7321 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     7381 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     7441 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     7501 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     7561 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7621 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7681 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     7741 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7801 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7861 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7921 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7981 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     8041 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     8101 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     8161 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtgagg gcctatttcc
     8221 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     8281 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     8341 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     8401 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgtaaa
     8461 atgctcctct gtttaggttt
//
