LOCUS       Exported                8465 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor ZNF197.
ACCESSION   .
VERSION     .
KEYWORDS    pZNF197.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8465)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8465)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8465
                     /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          691..918
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      930..939
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             939..1004
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1011..1031
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1056..5156
                     /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             5157..5204
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5232..5249)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5238..5445
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5454..5594
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5454..5583
                     /label=AAV2 ITR
     rep_origin      5669..6124
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5756..5775)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5966..5987
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6141..6160)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6260..6282
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6320..6338)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6406..6510
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6511..7371
                     /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(6729..6748)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7542..8130
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8031..8050
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8192..8432
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8192..8212
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8363..8382
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8462..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 cggggggcgg ggcggggcga ggcggagagg tgcggcggca
      601 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg
      661 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg
      721 ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
      781 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag caagaggtaa
      841 gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga gcacctgcct
      901 gaaatcactt tttttcaggt tggaccggtg ccaccatgga ctataaggac cacgacggag
      961 actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga
     1021 agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg
     1081 acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca
     1141 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag
     1201 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa
     1261 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
     1321 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
     1381 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
     1441 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
     1501 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
     1561 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc
     1621 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
     1681 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
     1741 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc
     1801 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
     1861 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
     1921 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
     1981 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
     2041 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
     2101 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg
     2161 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
     2221 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
     2281 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
     2341 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
     2401 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
     2461 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga
     2521 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
     2581 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg
     2641 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg
     2701 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
     2761 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct
     2821 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg
     2881 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag
     2941 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc
     3001 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca
     3061 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca
     3121 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga
     3181 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca
     3241 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag
     3301 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca
     3361 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag
     3421 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga
     3481 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca
     3541 tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg
     3601 actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg
     3661 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca
     3721 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg
     3781 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc
     3841 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga
     3901 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt
     3961 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga
     4021 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt
     4081 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg
     4141 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga
     4201 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg
     4261 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc
     4321 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta
     4381 tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga
     4441 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg
     4501 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg
     4561 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag
     4621 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc
     4681 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct
     4741 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg
     4801 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca
     4861 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag
     4921 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca
     4981 tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca
     5041 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc
     5101 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa
     5161 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa ttcctagagc
     5221 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     5281 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     5341 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     5401 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
     5461 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     5521 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     5581 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5641 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     5701 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     5761 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5821 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5881 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5941 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     6001 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     6061 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     6121 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     6181 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     6241 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     6301 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     6361 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     6421 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     6481 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6541 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6601 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6661 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6721 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6781 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6841 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6901 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6961 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     7021 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     7081 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     7141 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     7201 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     7261 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     7321 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     7381 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7441 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7501 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7561 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7621 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7681 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7741 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7801 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7861 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7921 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7981 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     8041 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     8101 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     8161 ctggcctttt gctggccttt tgctcacatg tgagggccta tttcccatga ttccttcata
     8221 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     8281 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     8341 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     8401 tttcttggct ttatatatct tgtggaaagg acgaaacacc gctggatgtg tctgtttctc
     8461 ggttt
//
