LOCUS       Exported                8490 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor ZNF165.
ACCESSION   .
VERSION     .
KEYWORDS    pZNF165.2.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8490)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8490)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8490
                     /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..583
                     /estimated_length=12
     intron          716..943
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      955..964
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             964..1029
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1036..1056
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1081..5181
                     /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             5182..5229
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5257..5274)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5263..5470
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5479..5619
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5479..5608
                     /label=AAV2 ITR
     rep_origin      5694..6149
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5781..5800)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5991..6012
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6166..6185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6285..6307
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6345..6363)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6431..6535
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6536..7396
                     /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(6754..6773)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7567..8155
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8056..8075
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8217..8457
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8217..8237
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8388..8407
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8487..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 nnnggcgngg ggcggggcgg
      601 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
      661 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt
      721 cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
      781 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
      841 ctgtaattag ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat
      901 gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc
      961 atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat
     1021 gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc
     1081 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
     1141 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
     1201 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
     1261 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
     1321 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
     1381 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
     1441 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
     1501 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     1561 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     1621 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     1681 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     1741 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
     1801 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     1861 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     1921 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     1981 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     2041 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     2101 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     2161 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     2221 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     2281 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     2341 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     2401 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     2461 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     2521 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     2581 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     2641 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     2701 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     2761 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     2821 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     2881 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     2941 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     3001 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     3061 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     3121 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     3181 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     3241 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     3301 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     3361 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     3421 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     3481 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     3541 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     3601 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     3661 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     3721 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     3781 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     3841 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     3901 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     3961 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     4021 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     4081 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     4141 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     4201 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     4261 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     4321 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     4381 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     4441 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
     4501 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     4561 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     4621 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     4681 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     4741 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     4801 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     4861 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     4921 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     4981 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     5041 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
     5101 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     5161 ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa
     5221 aagaaaaagt aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     5281 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     5341 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     5401 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc
     5461 atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     5521 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     5581 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     5641 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     5701 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     5761 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     5821 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     5881 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     5941 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     6001 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     6061 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     6121 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     6181 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     6241 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     6301 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     6361 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     6421 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     6481 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6541 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6601 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6661 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6721 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     6781 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6841 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6901 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6961 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     7021 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     7081 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     7141 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     7201 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg
     7261 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     7321 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     7381 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     7441 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     7501 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     7561 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     7621 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     7681 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     7741 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     7801 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     7861 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7921 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     7981 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     8041 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     8101 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     8161 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtgagg
     8221 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     8281 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     8341 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     8401 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     8461 acaccgaggg aaaacctatt aatgtagttt
//
