LOCUS MLM3639. 7614 bp DNA circular SYN 01-JAN-1980 DEFINITION Plasmid for expressing 3xFLAG(R)-tagged S. pyogenes Cas9 from either the CMV promoter in vivo or the T7 promoter in vitro. ACCESSION . VERSION . KEYWORDS MLM3639. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7614) AUTHORS Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. TITLE Efficient genome editing in zebrafish using a CRISPR-Cas system. JOURNAL Nat. Biotechnol. 2013;31:227-9. PUBMED 23360964 REFERENCE 2 (bases 1 to 7614) AUTHORS Joung Lab / Addgene #42252 TITLE Direct Submission REFERENCE 3 (bases 1 to 7614) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat. Biotechnol."; date: "2013"; volume: "31"; pages: "227-9" COMMENT SGRef: number: 2; type: "Journal Article" COMMENT Linearize with PmeI for in vitro transcription. FEATURES Location/Qualifiers source 1..7614 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 132..335 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 377..395 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" CDS 409..4512 /label=Cas9 /note="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" CDS 4519..4539 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /translation="PKKKRKV" CDS 4546..4611 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=three tandem FLAG /note="3xFLAG" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4629..4646 /label=6xHis /note="6xHis affinity tag" polyA_signal 4675..4899 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(4970..4986) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(4994..5010) /label=lac operator /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(5018..5048) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(5063..5084) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(5372..5960) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6134..6991) /label=AmpR /note="beta-lactamase" promoter complement(6992..7096) /label=AmpR promoter enhancer 7366..7614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggataagaaa 421 tactcaatag gcttagatat cggcacaaat agcgtcggat gggcggtgat cactgatgaa 481 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa 541 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc 601 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag 661 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct 721 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat 781 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat 841 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt 901 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta 961 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt 1021 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat 1081 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg 1141 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta 1201 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat 1261 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat 1321 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc 1381 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca 1441 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat 1501 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat 1561 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc 1621 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga 1681 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg 1741 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg 1801 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa 1861 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat 1921 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg 1981 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag 2041 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta 2101 aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa 2161 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa 2221 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc 2281 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat 2341 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga 2401 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa 2461 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt 2521 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt 2581 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt 2641 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca 2701 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc 2761 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact 2821 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg 2881 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatcacat tgttccacaa 2941 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt 3001 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga 3061 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa 3121 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact 3181 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat 3241 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct 3301 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc 3361 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt 3421 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag 3481 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac 3541 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa 3601 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc 3661 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga 3721 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa 3781 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta 3841 gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta 3901 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct 3961 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt 4021 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat 4081 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag 4141 ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat 4201 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat 4261 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa 4321 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt 4381 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat 4441 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag 4501 ctaggaggtg acggttctcc caagaagaag aggaaagtct cgagcgacta caaagaccat 4561 gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa ggctgcagga 4621 tgaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc 4681 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 4741 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 4801 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 4861 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 4921 gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt 4981 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 5041 agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 5101 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 5161 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 5221 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 5281 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 5341 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5401 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5461 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5521 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5581 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5641 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5701 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5761 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5821 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5881 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5941 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6001 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 6061 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 6121 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 6181 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 6241 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 6301 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 6361 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 6421 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 6481 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 6541 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 6601 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 6661 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 6721 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 6781 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 6841 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6901 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6961 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 7021 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 7081 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag 7141 atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt 7201 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa 7261 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta 7321 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga 7381 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 7441 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 7501 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 7561 aatgggtgga ctatttacgg taaactgccc acttggcagt acatcaagtg tatc //