LOCUS       pBS-Hsp70-Cas9.        8699 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial plasmid encoding S. pyogenes Cas9 under control of the 
            Drosophila hsp70 promoter and poly(A) signal.
ACCESSION   .
VERSION     .
KEYWORDS    pBS-Hsp70-Cas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8699)
  AUTHORS   O'Connor-Giles Lab / Addgene #46294
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8699)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     This plasmid sequence was inferred from the cloning procedure 
            described on the Addgene website. The orientation of the NotI insert
            is unknown.
FEATURES             Location/Qualifiers
     source          1..8699
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(6..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        692..1144
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     CDS             1160..1162
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             1163..1228
                     /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             1235..1255
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             1280..5380
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             5381..5428
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=bipartite nuclear localization signal from
                     nucl
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5508..6380
                     /label=hsp70 poly(A)
                     /note="polyadenlyation signal from the Drosophila
                     melanogaster hsp70Ab gene"
     primer_bind     6418..6434
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6468..6484)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(6514..6532)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6553..6569)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(6577..6593)
                     /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(6601..6631)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6646..6667)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6955..7543)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7717..8574)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8575..8679)
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgcggaa ttcccatccc cctagaatcc caaaacaaac tggttattgt
      721 ggtaggtcat ttgtttggca gaaagaaaac tcgagaaatt tctctggccg ttattcgtta
      781 ttctctcttt tctttttggg tctctccctc tctgcactaa tgctctctca ctctgtcaca
      841 cagtaaacgg catactgctc tcgttggttc gagagagcgc gcctcgaatg ttcgcgaaaa
      901 gagcgccgga gtataaatag aggcgcttcg tctacggagc gacaattcaa ttcaaacaag
      961 caaagtgaac acgtcgctaa gcgaaagcta agcaaataaa caagcgcagc tgaacaagct
     1021 aaacaatctg cagtaaagtg caagttaaag tgaatcaatt aaaagtaacc agcaaccaag
     1081 taaatcaact gcaactactg aaatctgcca agaagtaatt attgaataca agaagagaac
     1141 tctgggggat ctgatcgata tggactataa ggaccacgac ggagactaca aggatcatga
     1201 tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat
     1261 ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc
     1321 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct
     1381 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag
     1441 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg
     1501 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga
     1561 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg
     1621 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat
     1681 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta
     1741 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa
     1801 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct
     1861 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag
     1921 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa
     1981 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa
     2041 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct
     2101 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa
     2161 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc
     2221 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct
     2281 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag
     2341 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt
     2401 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag
     2461 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca
     2521 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga
     2581 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct
     2641 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc
     2701 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat
     2761 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta
     2821 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag
     2881 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac
     2941 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt
     3001 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca
     3061 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat
     3121 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg
     3181 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag
     3241 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc
     3301 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
     3361 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
     3421 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
     3481 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
     3541 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
     3601 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
     3661 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     3721 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     3781 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
     3841 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
     3901 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     3961 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     4021 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     4081 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     4141 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     4201 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
     4261 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt
     4321 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
     4381 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     4441 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     4501 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     4561 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     4621 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
     4681 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     4741 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     4801 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     4861 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     4921 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     4981 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
     5041 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
     5101 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
     5161 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
     5221 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag
     5281 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
     5341 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa
     5401 aaaggccggc caggcaaaaa agaaaaagta atctagagat cttccatacc taccagttct
     5461 gcgcctgcag caatggcaac aacgttgccc ggatcgaggt cgactaaggc caaagagtct
     5521 aatttttgtt catcaatggg ttataacata tgggttatat tataagtttg ttttaagttt
     5581 ttgagactga taagaatgtt tcgatcgaat attccataga acaacaatag tattacctaa
     5641 ttaccaagtc ttaatttagc aaaaatgtta ttgcttatag aaaaaataaa ttatttattt
     5701 gaaatttaaa gtcaacttgt catttaatgt cttgtagact tttgaaagtc ttacgataca
     5761 attagtatct aatatacatg ggttcattct acattctata ttagtgatga tttctttagc
     5821 tagtaataca ttttaattat attcggcttt gatgattttc tgattttttc cgaacggatt
     5881 ttcgtagacc ctttcgatct cataatggct cattttattg cgatggacgg tcaggagagc
     5941 tccacttttg aatttctgtt cgcagacacc gcatttgtag cacatagccg ggacatccgg
     6001 tttggggaga ttttccagtc tctgttgcaa ttggttttcg ggaatgcgtt gcaggcgcat
     6061 acgctctata tcctccgaac ggcgctggtt gaccctagca tttacataag gatcagcagc
     6121 aaaatttgcc tctacttcat tgcccggaat cacagcaatc agatgtccct ttcggttacg
     6181 atggatattc aggtgcgaac cgcacacaaa gctctcgccg cacactccac actgatatgg
     6241 tcgctcgccc gtgtggcgcc gcatatggat cttaaggtcg ttggactgca caaagctctt
     6301 gctgcacatt ttgcaggagt acggcctttg acccgtgtgc aatcgcatgt gtcgcgccag
     6361 cttgttctgc gaaataaact aacgggaatt cctgcagccc gggggatccg cggccgctct
     6421 agaactagtg gatcccccgg gctgcaggaa ttcgatatca agcttatcga taccgtcgac
     6481 ctcgaggggg ggcccggtac ccagcttttg ttccctttag tgagggttaa tttcgagctt
     6541 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     6601 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     6661 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     6721 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     6781 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     6841 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     6901 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     6961 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     7021 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     7081 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     7141 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     7201 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     7261 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     7321 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     7381 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     7441 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     7501 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     7561 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     7621 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     7681 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     7741 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7801 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7861 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7921 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7981 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     8041 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     8101 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     8161 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     8221 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     8281 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     8341 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     8401 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     8461 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     8521 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     8581 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     8641 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgc
//
