LOCUS       Exported                8149 bp ds-DNA     circular SYN 12-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 8149)
  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 8149)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8149
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        841..1293
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     1240..1261
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     CDS             1312..1377
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1384..1404
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1429..5529
                     /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             5530..5577
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5657..6529
                     /label=hsp70 poly(A)
                     /note="polyadenlyation signal from the Drosophila 
                     melanogaster hsp70Ab gene"
     CDS             complement(6742..7536)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6853..6872)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7463..7482
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(8003..8024)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tactgaacgg tgatccccac cggaattgcg gccgcggaat tcccatcccc
      841 ctagaatccc aaaacaaact ggttattgtg gtaggtcatt tgtttggcag aaagaaaact
      901 cgagaaattt ctctggccgt tattcgttat tctctctttt ctttttgggt ctctccctct
      961 ctgcactaat gctctctcac tctgtcacac agtaaacggc atactgctct cgttggttcg
     1021 agagagcgcg cctcgaatgt tcgcgaaaag agcgccggag tataaataga ggcgcttcgt
     1081 ctacggagcg acaattcaat tcaaacaagc aaagtgaaca cgtcgctaag cgaaagctaa
     1141 gcaaataaac aagcgcagct gaacaagcta aacaatctgc agtaaagtgc aagttaaagt
     1201 gaatcaatta aaagtaacca gcaaccaagt aaatcaactg caactactga aatctgccaa
     1261 gaagtaatta ttgaatacaa gaagagaact ctgggggatc tgatcgatat ggactataag
     1321 gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg
     1381 gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac
     1441 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     1501 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     1561 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     1621 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     1681 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     1741 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     1801 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     1861 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     1921 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     1981 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     2041 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     2101 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     2161 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     2221 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     2281 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     2341 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     2401 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     2461 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     2521 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     2581 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     2641 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     2701 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     2761 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     2821 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     2881 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     2941 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     3001 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     3061 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     3121 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     3181 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     3241 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     3301 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     3361 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     3421 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     3481 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     3541 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     3601 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     3661 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     3721 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     3781 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
     3841 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     3901 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
     3961 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     4021 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     4081 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     4141 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     4201 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     4261 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     4321 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     4381 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     4441 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     4501 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     4561 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     4621 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     4681 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     4741 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     4801 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     4861 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
     4921 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     4981 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     5041 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     5101 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     5161 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     5221 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
     5281 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
     5341 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
     5401 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
     5461 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg
     5521 ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa
     5581 tctagagatc ttccatacct accagttctg cgcctgcagc aatggcaaca acgttgcccg
     5641 gatcgaggtc gactaaggcc aaagagtcta atttttgttc atcaatgggt tataacatat
     5701 gggttatatt ataagtttgt tttaagtttt tgagactgat aagaatgttt cgatcgaata
     5761 ttccatagaa caacaatagt attacctaat taccaagtct taatttagca aaaatgttat
     5821 tgcttataga aaaaataaat tatttatttg aaatttaaag tcaacttgtc atttaatgtc
     5881 ttgtagactt ttgaaagtct tacgatacaa ttagtatcta atatacatgg gttcattcta
     5941 cattctatat tagtgatgat ttctttagct agtaatacat tttaattata ttcggctttg
     6001 atgattttct gattttttcc gaacggattt tcgtagaccc tttcgatctc ataatggctc
     6061 attttattgc gatggacggt caggagagct ccacttttga atttctgttc gcagacaccg
     6121 catttgtagc acatagccgg gacatccggt ttggggagat tttccagtct ctgttgcaat
     6181 tggttttcgg gaatgcgttg caggcgcata cgctctatat cctccgaacg gcgctggttg
     6241 accctagcat ttacataagg atcagcagca aaatttgcct ctacttcatt gcccggaatc
     6301 acagcaatca gatgtccctt tcggttacga tggatattca ggtgcgaacc gcacacaaag
     6361 ctctcgccgc acactccaca ctgatatggt cgctcgcccg tgtggcgccg catatggatc
     6421 ttaaggtcgt tggactgcac aaagctcttg ctgcacattt tgcaggagta cggcctttga
     6481 cccgtgtgca atcgcatgtg tcgcgccagc ttgttctgcg aaataaacta acgggaattc
     6541 ctgcagcccg ggggatccgc ggccgcccgg ggtgggcgaa gaactccagc atgagatccc
     6601 cgcgctggag gatcatccag ccggcgtccc ggaaaacgat tccgaagccc aacctttcat
     6661 agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt gggcgtcgct tggtcggtca
     6721 tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg
     6781 ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc
     6841 aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc
     6901 cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa
     6961 gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct
     7021 ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac
     7081 aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa
     7141 tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac
     7201 tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag
     7261 cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt
     7321 cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg caccggacag
     7381 gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc
     7441 agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc
     7501 cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc
     7561 ttgatcagat cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt
     7621 tactttgcag ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccggttcgct
     7681 tgctgtccat aaaaccgccc agtctagcta tcgccatgta agcccactgc aagctacctg
     7741 ctttctcttt gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg
     7801 gggtcagcac cgtttctgcg gactggcttt ctacgtgttc cgcttccttt agcagccctt
     7861 gcgccctgag tgcttgcggc agcgtgaagc taattcatgg ttaaattttt gttaaatcag
     7921 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagcc
     7981 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     8041 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggccggatca gcttatgcgg
     8101 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgct
//
