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
//
