LOCUS       Exported                8469 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR plasmid for expression of Cas9 and gRNA targeting human 
            transcription factor ZNF311.
ACCESSION   .
VERSION     .
KEYWORDS    pZNF311.1.0-gDNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8469)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 8469)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8469
                     /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          696..923
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      935..944
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             944..1009
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1016..1036
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1061..5161
                     /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             5162..5209
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5237..5254)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5243..5450
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5459..5599
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5459..5588
                     /label=AAV2 ITR
     rep_origin      5674..6129
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5761..5780)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5971..5992
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6146..6165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6265..6287
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6325..6343)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6411..6515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6516..7376
                     /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(6734..6753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7547..8135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8036..8055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8197..8437
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8197..8217
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8368..8387
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8466..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 gggggggcgg ggcgaggcgg agaggtgcgg
      601 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
      661 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
      721 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
      781 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga
      841 ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc
      901 tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata aggaccacga
      961 cggagactac aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa
     1021 gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg
     1081 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     1141 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     1201 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     1261 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     1321 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     1381 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     1441 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     1501 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     1561 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     1621 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     1681 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     1741 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     1801 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     1861 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     1921 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     1981 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     2041 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     2101 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     2161 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     2221 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     2281 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     2341 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     2401 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     2461 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     2521 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     2581 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     2641 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     2701 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     2761 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     2821 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     2881 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     2941 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     3001 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     3061 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     3121 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     3181 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     3241 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     3301 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     3361 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     3421 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     3481 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     3541 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
     3601 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     3661 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     3721 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     3781 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     3841 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     3901 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     3961 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     4021 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
     4081 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     4141 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     4201 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
     4261 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     4321 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     4381 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     4441 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
     4501 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     4561 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     4621 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     4681 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
     4741 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     4801 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     4861 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     4921 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     4981 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
     5041 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
     5101 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
     5161 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcct
     5221 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     5281 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     5341 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     5401 caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag
     5461 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     5521 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     5581 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     5641 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     5701 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     5761 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     5821 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     5881 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5941 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     6001 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     6061 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     6121 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     6181 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     6241 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     6301 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     6361 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     6421 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     6481 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     6541 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     6601 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     6661 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6721 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6781 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6841 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6901 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6961 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     7021 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     7081 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     7141 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     7201 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     7261 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     7321 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     7381 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     7441 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     7501 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     7561 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     7621 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     7681 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     7741 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     7801 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     7861 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7921 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7981 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     8041 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     8101 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     8161 ggttcctggc cttttgctgg ccttttgctc acatgtgagg gcctatttcc catgattcct
     8221 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     8281 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     8341 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     8401 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgtgcc tgagtataac
     8461 gattcgttt
//
