LOCUS Exported 5732 bp ds-DNA linear SYN 28-MAY-2021 DEFINITION The codon-optimized Cas9 nuclease under the control of the Drosophila hsp70 promoter used in Gratz, et al. (2013). Plasmid is low copy number.. ACCESSION . VERSION . KEYWORDS pHsp70-Cas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5732) AUTHORS Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O'Connor-Giles KM TITLE Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. JOURNAL Genetics. 2013 May 24. PUBMED 23709638 REFERENCE 2 (bases 1 to 5732) AUTHORS . TITLE Direct Submission JOURNAL Exported May 28, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5732 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 7..459 /label=hsp70 promoter /note="Drosophila melanogaster hsp70Bb promoter" primer_bind 406..427 /label=pCasper-hs /note="Drosophila Hsp70 promoter, forward primer" CDS 478..543 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 550..570 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 595..4695 /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 4696..4743 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" polyA_signal 4823..5695 /label=hsp70 poly(A) /note="polyadenlyation signal from the Drosophila melanogaster hsp70Ab gene" ORIGIN 1 atccccctag aatcccaaaa caaactggtt attgtggtag gtcatttgtt tggcagaaag 61 aaaactcgag aaatttctct ggccgttatt cgttattctc tcttttcttt ttgggtctct 121 ccctctctgc actaatgctc tctcactctg tcacacagta aacggcatac tgctctcgtt 181 ggttcgagag agcgcgcctc gaatgttcgc gaaaagagcg ccggagtata aatagaggcg 241 cttcgtctac ggagcgacaa ttcaattcaa acaagcaaag tgaacacgtc gctaagcgaa 301 agctaagcaa ataaacaagc gcagctgaac aagctaaaca atctgcagta aagtgcaagt 361 taaagtgaat caattaaaag taaccagcaa ccaagtaaat caactgcaac tactgaaatc 421 tgccaagaag taattattga atacaagaag agaactctgg gggatctgat cgatatggac 481 tataaggacc acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat 541 aagatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag 601 aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac 661 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc 721 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg 781 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa 841 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag 901 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg 961 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg 1021 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag 1081 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag 1141 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc 1201 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa 1261 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc 1321 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa 1381 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc 1441 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc 1501 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag 1561 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg 1621 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt 1681 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg 1741 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg 1801 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg 1861 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc 1921 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc 1981 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac 2041 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc 2101 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag 2161 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag 2221 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag 2281 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg 2341 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac 2401 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg 2461 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc 2521 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc 2581 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg 2641 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc 2701 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac 2761 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg 2821 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg 2881 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg 2941 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac 3001 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac 3061 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct 3121 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac 3181 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg 3241 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc 3301 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa 3361 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac 3421 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg 3481 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac 3541 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag 3601 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc 3661 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg 3721 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc 3781 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 3841 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 3901 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag 3961 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg 4021 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 4081 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 4141 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 4201 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga 4261 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 4321 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 4381 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 4441 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 4501 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc 4561 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg 4621 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 4681 cagctgggag gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa 4741 aagtaatcta gagatcttcc atacctacca gttctgcgcc tgcagcaatg gcaacaacgt 4801 tgcccggatc gaggtcgact aaggccaaag agtctaattt ttgttcatca atgggttata 4861 acatatgggt tatattataa gtttgtttta agtttttgag actgataaga atgtttcgat 4921 cgaatattcc atagaacaac aatagtatta cctaattacc aagtcttaat ttagcaaaaa 4981 tgttattgct tatagaaaaa ataaattatt tatttgaaat ttaaagtcaa cttgtcattt 5041 aatgtcttgt agacttttga aagtcttacg atacaattag tatctaatat acatgggttc 5101 attctacatt ctatattagt gatgatttct ttagctagta atacatttta attatattcg 5161 gctttgatga ttttctgatt ttttccgaac ggattttcgt agaccctttc gatctcataa 5221 tggctcattt tattgcgatg gacggtcagg agagctccac ttttgaattt ctgttcgcag 5281 acaccgcatt tgtagcacat agccgggaca tccggtttgg ggagattttc cagtctctgt 5341 tgcaattggt tttcgggaat gcgttgcagg cgcatacgct ctatatcctc cgaacggcgc 5401 tggttgaccc tagcatttac ataaggatca gcagcaaaat ttgcctctac ttcattgccc 5461 ggaatcacag caatcagatg tccctttcgg ttacgatgga tattcaggtg cgaaccgcac 5521 acaaagctct cgccgcacac tccacactga tatggtcgct cgcccgtgtg gcgccgcata 5581 tggatcttaa ggtcgttgga ctgcacaaag ctcttgctgc acattttgca ggagtacggc 5641 ctttgacccg tgtgcaatcg catgtgtcgc gccagcttgt tctgcgaaat aaactaacgg 5701 gaattcctgc agcccggggg atccgcggcc gc //