LOCUS Exported 8509 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION BRD4 tagging CRISPR. ACCESSION . VERSION . KEYWORDS BRD4-N CRISPR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8509) AUTHORS Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano R, Nakaoka H, Fukui K, Gamo K, Tominari Y, Takeuchi H, Saga Y, Hayashi KI, Kanemaki MT. TITLE The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice. JOURNAL Nat Commun (2020) 11(1):5701 REFERENCE 2 (bases 1 to 8509) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8509 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 271..346 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 443..728 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 730..1007 /label=chicken beta-actin promoter intron 1008..1236 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1248..1257 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1257..1322 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1329..1349 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1374..5474 /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 5475..5522 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5550..5567) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5556..5763 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5772..5912 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5772..5901 /label=AAV2 ITR rep_origin 5987..6442 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6074..6093) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6284..6305 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6459..6478) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6578..6600 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6638..6656) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6724..6828 /gene="bla" /label=AmpR promoter CDS 6829..7689 /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(7047..7066) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7860..8448 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8349..8368 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg atgtctgcgg agagcggccc gttttagagc tagaaatagc aagttaaaat 301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga 361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca 421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac 481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac 541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc 661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 841 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 901 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga 1021 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga 1081 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat 1141 tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat 1201 tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact 1261 ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata 1321 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga 1381 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg 1441 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca 1501 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc 1561 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag 1621 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt 1681 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg 1741 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg 1801 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt 1861 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc 1921 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca 1981 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa 2041 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc 2101 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac 2161 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg 2221 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg 2281 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga 2341 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc 2401 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg 2461 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg 2521 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga 2581 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc 2641 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc 2701 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct 2761 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca 2821 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca 2881 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc 2941 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc 3001 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc 3061 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg 3121 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca 3181 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga 3241 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg 3301 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc 3361 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga 3421 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct 3481 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca 3541 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg 3601 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg 3661 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga 3721 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca 3781 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg 3841 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc 3901 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc 3961 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc 4021 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg 4081 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa 4141 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg 4201 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt 4261 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg 4321 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc 4381 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca 4441 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga 4501 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg 4561 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc 4621 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg 4681 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga 4741 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc 4801 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc 4861 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag 4921 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt 4981 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa 5041 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga 5101 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc 5161 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg 5221 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag 5281 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct 5341 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg 5401 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc 5461 agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa 5521 agtaagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 5581 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 5641 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 5701 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg 5761 ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc 5821 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc 5881 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac 5941 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 6001 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 6061 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 6121 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 6181 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 6241 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 6301 actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg 6361 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 6421 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 6481 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 6541 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 6601 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 6661 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 6721 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 6781 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 6841 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 6901 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 6961 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 7021 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 7081 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 7141 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 7201 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 7261 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 7321 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 7381 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 7441 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 7501 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt 7561 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 7621 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 7681 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 7741 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 7801 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 7861 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 7921 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 7981 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 8041 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 8101 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 8161 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 8221 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 8281 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 8341 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 8401 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 8461 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt //