LOCUS       Exported                8474 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor ZNF75A.
ACCESSION   .
VERSION     .
KEYWORDS    pZNF75A.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8474)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8474)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8474
                     /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          700..927
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      939..948
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             948..1013
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1020..1040
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1065..5165
                     /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             5166..5213
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5241..5258)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5247..5454
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5463..5603
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5463..5592
                     /label=AAV2 ITR
     rep_origin      5678..6133
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5765..5784)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5975..5996
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6150..6169)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6269..6291
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6329..6347)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6415..6519
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6520..7380
                     /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(6738..6757)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7551..8139
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8040..8059
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8201..8441
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8201..8221
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8372..8391
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8471..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 gcggggcgag gcggagaggt
      601 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
      661 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt
      721 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
      781 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagctgagc
      841 aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctggag
      901 cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc caccatggac tataaggacc
      961 acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc
     1021 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca
     1081 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     1141 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     1201 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
     1261 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     1321 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     1381 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     1441 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     1501 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     1561 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     1621 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     1681 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     1741 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     1801 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     1861 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     1921 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     1981 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     2041 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     2101 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     2161 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     2221 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     2281 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     2341 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     2401 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     2461 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     2521 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     2581 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     2641 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     2701 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     2761 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     2821 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     2881 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     2941 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     3001 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     3061 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     3121 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     3181 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     3241 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     3301 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     3361 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     3421 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     3481 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     3541 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     3601 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     3661 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     3721 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     3781 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     3841 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     3901 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     3961 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     4021 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     4081 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     4141 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     4201 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     4261 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     4321 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     4381 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     4441 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     4501 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     4561 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     4621 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     4681 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     4741 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     4801 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     4861 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     4921 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     4981 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     5041 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     5101 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     5161 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagtaagaat
     5221 tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     5281 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     5341 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     5401 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc
     5461 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     5521 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5581 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     5641 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     5701 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     5761 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     5821 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     5881 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5941 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     6001 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     6061 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     6121 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6181 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     6241 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     6301 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     6361 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     6421 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6481 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6541 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6601 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6661 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6721 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6781 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6841 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6901 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6961 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     7021 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     7081 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     7141 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     7201 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     7261 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     7321 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     7381 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7441 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7501 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7561 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7621 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7681 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7741 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7801 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7861 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7921 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7981 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     8041 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     8101 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     8161 ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat ttcccatgat
     8221 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
     8281 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     8341 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     8401 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gacaagcttc
     8461 tcttcacagg gttt
//
