LOCUS Exported 8494 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CRISPR plasmid for expression of Cas9 and gRNA targeting human transcription factor MYNN. ACCESSION . VERSION . KEYWORDS pMYNN.1.0-gDNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8494) TITLE White lab ENCODE plasmids to tag human transcription factors with GFP REFERENCE 2 (bases 1 to 8494) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8494 /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 574..586 /estimated_length=13 intron 720..947 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 959..968 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 968..1033 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1040..1060 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1085..5185 /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 5186..5233 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5261..5278) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5267..5474 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5483..5623 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5483..5612 /label=AAV2 ITR rep_origin 5698..6153 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5785..5804) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5995..6016 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6170..6189) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6289..6311 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6349..6367) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6435..6539 /gene="bla" /label=AmpR promoter CDS 6540..7400 /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(6758..6777) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7571..8159 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8060..8079 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 8221..8461 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8221..8241 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8392..8411 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8491..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 gggnnnnnnn nnnnnngggc gaggggcggg 601 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 661 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg 721 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc 781 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 841 cgggctgtaa ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat 901 taatgtttaa ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc 961 caccatggac tataaggacc acgacggaga ctacaaggat catgatattg attacaaaga 1021 cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc 1081 agccgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt 1141 gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg 1201 gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga 1261 ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg 1321 ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag 1381 actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg 1441 caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa 1501 gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca 1561 catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga 1621 cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc 1681 catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag 1741 acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa 1801 cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga 1861 ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc 1921 ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat 1981 cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc 2041 tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg 2101 gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc 2161 cggctacatt gacggcggag ccagccagga agagttctac aagttcatca agcccatcct 2221 ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg 2281 gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca 2341 cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat 2401 cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag 2461 cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga 2521 agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa 2581 gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt 2641 gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct 2701 gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac 2761 cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat 2821 ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat 2881 tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt 2941 gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc 3001 ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg 3061 caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct 3121 ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga 3181 cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct 3241 gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac 3301 agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt 3361 gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag 3421 aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc 3481 cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg 3541 ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca 3601 tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag 3661 cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa 3721 gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct 3781 gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca 3841 gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa 3901 cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc 3961 caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa 4021 ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa 4081 gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa 4141 gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag 4201 caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg 4261 gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt 4321 tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt 4381 gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat 4441 cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc 4501 ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt 4561 gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga 4621 ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa 4681 gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact 4741 gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag 4801 ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga 4861 acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt 4921 gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa 4981 gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc 5041 ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa 5101 agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat 5161 cgacctgtct cagctgggag gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc 5221 aaaaaagaaa aagtaagaat tcctagagct cgctgatcag cctcgactgt gccttctagt 5281 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 5341 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 5401 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc 5461 aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg 5521 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc 5581 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat 5641 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg 5701 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 5761 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 5821 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 5881 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat 5941 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 6001 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag 6061 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg 6121 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct 6181 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 6241 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 6301 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 6361 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 6421 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 6481 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 6541 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 6601 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 6661 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 6721 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 6781 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 6841 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 6901 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 6961 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 7021 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 7081 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 7141 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 7201 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc 7261 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 7321 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 7381 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 7441 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 7501 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 7561 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 7621 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 7681 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 7741 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 7801 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 7861 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 7921 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 7981 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 8041 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 8101 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 8161 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 8221 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 8281 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 8341 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 8401 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 8461 cgaaacaccg acaattatac tgactttgta gttt //