LOCUS       pCas-Guide-Nicka        8033 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Mammalian vector for co-expressing a guide RNA (gRNA) together with 
            the D10A nickase mutant of Cas9, for genome editing using the 
            CRISPR/Cas9 system.
ACCESSION   .
VERSION     .
KEYWORDS    pCas-Guide-Nickase
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8033)
  AUTHORS   OriGene
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8033)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        90..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        384..459
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      460..465
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     enhancer        511..890
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        891..1094
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        1120..1138
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1197..5300
                     /label=Cas9(D10A)
                     /note="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             5301..5321
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             5328..5348
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             5370..5399
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
     CDS             5418..5441
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     primer_bind     complement(5461..5477)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    5488..6110
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     rep_origin      complement(6231..6819)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6997..7854)
                     /label=AmpR
                     /note="beta-lactamase"
     primer_bind     7969..7985
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gaattcccca gtggaaagac gcgcaggcaa aacgcaccac gtgacggagc gtgaccgcgc
       61 gccgagcgcg cgccaaggtc gggcaggaag agggcctatt tcccatgatt ccttcatatt
      121 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
      181 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
      241 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
      301 tcttgggttt atatatcttg tggaaaggac gcgggatcca ctggaccagg cagcagcgtc
      361 agaagacttt tttggaacgt ctcgttttag agctagaaat agcaagttaa aataaggcta
      421 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttggtg tacatttata
      481 ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt tattaatagt
      541 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      601 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      661 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      721 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta
      781 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg
      841 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      901 tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      961 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     1021 gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     1081 atataagcag agctcgttta gtgaaccgtc agaattttgt aatacgactc actatagggc
     1141 ggccgggaat tcgtcgactg gaaccggtac cgaggagatc tgccgccgcg atcgccatgg
     1201 ataagaaata ctcaatagga ctggctattg gcacaaatag cgtcggatgg gctgtgatca
     1261 ctgatgaata taaggttcct tctaaaaagt tcaaggttct gggaaataca gaccgccaca
     1321 gtatcaaaaa aaatcttata ggggctcttc tgtttgacag tggagagaca gccgaagcta
     1381 ctagactcaa acggacagct aggagaaggt atacaagacg gaagaatagg atttgttatc
     1441 tccaggagat tttttcaaat gagatggcca aagtggatga tagtttcttt catagacttg
     1501 aagagtcttt tttggtggaa gaagacaaga agcatgaaag acatcctatt tttggaaata
     1561 tagtggatga agttgcttat cacgagaaat atccaactat ctatcatctg agaaaaaaat
     1621 tggtggattc tactgataaa gccgatttgc gcctgatcta tttggccctg gcccacatga
     1681 ttaagtttag aggtcatttt ttgattgagg gcgatctgaa tcctgataat agtgatgtgg
     1741 acaaactgtt tatccagttg gtgcaaacct acaatcaact gtttgaagaa aaccctatta
     1801 acgcaagtgg agtggatgct aaagccattc tttctgcaag attgagtaaa tcaagaagac
     1861 tggaaaatct cattgctcag ctccccggtg agaagaaaaa tggcctgttt gggaatctca
     1921 ttgctttgtc attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg
     1981 ctaaactcca gctttcaaaa gatacttacg atgatgatct ggataatctg ttggctcaaa
     2041 ttggagatca atatgctgat ttgtttttgg cagctaagaa tctgtcagat gctattctgc
     2101 tttcagacat cctgagagtg aatactgaaa taactaaggc tcccctgtca gcttcaatga
     2161 ttaaacgcta cgatgaacat catcaagact tgactcttct gaaagccctg gttagacaac
     2221 aacttccaga aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt
     2281 atattgatgg cggcgcaagc caagaagaat tttataaatt tatcaaacca attctggaaa
     2341 aaatggatgg tactgaggaa ctgttggtga aactgaatag agaagatttg ctgcgcaagc
     2401 aacggacctt tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta
     2461 ttttgagaag acaagaagac ttttatccat ttctgaaaga caatagagag aagattgaaa
     2521 aaatcttgac ttttaggatt ccttattatg ttggtccatt ggccagaggc aatagtaggt
     2581 ttgcatggat gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg
     2641 tcgataaagg tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc
     2701 ttccaaatga aaaagtgctg ccaaaacata gtttgcttta tgagtatttt accgtttata
     2761 acgaattgac aaaggtcaaa tatgttactg aaggaatgag aaaaccagca tttctttcag
     2821 gtgaacagaa gaaagccatt gttgatctgc tcttcaaaac aaataggaaa gtgaccgtta
     2881 agcaactgaa agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag
     2941 gagttgaaga tagatttaat gcttcactgg gtacatacca tgatttgctg aaaattatta
     3001 aagataaaga ttttttggat aatgaagaaa atgaagacat cctggaggat attgttctga
     3061 cattgaccct gtttgaagat agggagatga ttgaggaaag acttaaaaca tacgctcacc
     3121 tctttgatga taaggtgatg aaacagctta aaagacgcag atatactggt tggggaaggt
     3181 tgtccagaaa attgattaat ggtattaggg ataagcaatc tggcaaaaca atactggatt
     3241 ttttgaaatc agatggtttt gccaatcgca attttatgca gctcatccat gatgatagtt
     3301 tgacatttaa agaagacatc caaaaagcac aagtgtctgg acaaggcgat agtctgcatg
     3361 aacatattgc aaatctggct ggtagccctg ctattaaaaa aggtattctc cagactgtga
     3421 aagttgttga tgaattggtc aaagtgatgg ggcggcataa gccagaaaat atcgttattg
     3481 aaatggcaag agaaaatcag acaactcaaa agggccagaa aaattccaga gagaggatga
     3541 aaagaatcga agaaggtatc aaagaactgg gaagtcagat tcttaaagag catcctgttg
     3601 aaaatactca attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagata
     3661 tgtatgtgga ccaagaactg gatattaata ggctgagtga ttatgatgtc gatcacattg
     3721 ttccacaaag tttccttaaa gacgattcaa tagacaataa ggtcctgacc aggtctgata
     3781 aaaatagagg taaatccgat aacgttccaa gtgaagaagt ggtcaaaaag atgaaaaact
     3841 attggagaca acttctgaac gccaagctga tcactcaaag gaagtttgat aatctgacca
     3901 aagctgaaag aggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg
     3961 ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta
     4021 aatacgatga aaatgataaa cttattagag aggttaaagt gattaccctg aaatctaaac
     4081 tggtttctga cttcagaaaa gatttccaat tctataaagt gagagagatt aacaattacc
     4141 atcatgccca tgatgcctat ctgaatgccg tcgttggaac tgctttgatt aagaaatatc
     4201 caaaacttga aagcgagttt gtctatggtg attataaagt ttatgatgtt aggaaaatga
     4261 ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa gtatttcttt tactctaata
     4321 tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc
     4381 tgatcgaaac taatggggaa actggagaaa ttgtctggga taaagggaga gattttgcca
     4441 cagtgcgcaa agtgttgtcc atgccccaag tcaatatcgt caagaaaaca gaagtgcaga
     4501 caggcggatt ctctaaggag tcaattctgc caaaaagaaa ttccgacaag ctgattgcta
     4561 ggaaaaaaga ctgggaccca aaaaaatatg gtggttttga tagtccaacc gtggcttatt
     4621 cagtcctggt ggttgctaag gtggaaaaag ggaaatccaa gaagctgaaa tccgttaaag
     4681 agctgctggg gatcacaatt atggaaagaa gttcctttga aaaaaatccc attgactttc
     4741 tggaagctaa aggatataag gaagttaaaa aagacctgat cattaaactg cctaaatata
     4801 gtctttttga gctggaaaac ggtaggaaac ggatgctggc tagtgccgga gaactgcaaa
     4861 aaggaaatga gctggctctg ccaagcaaat atgtgaattt tctgtatctg gctagtcatt
     4921 atgaaaagtt gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc
     4981 ataagcatta tctggatgag attattgagc aaatcagtga attttctaag agagttattc
     5041 tggcagatgc caatctggat aaagttctta gtgcatataa caaacataga gacaaaccaa
     5101 taagagaaca agcagaaaat atcattcatc tgtttacctt gaccaatctt ggagcacccg
     5161 ctgcttttaa atactttgat acaacaattg ataggaaaag atatacctct acaaaagaag
     5221 ttctggatgc cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt
     5281 tgagtcagct gggaggtgac cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc
     5341 ggaaagtgga cacgcgtacg cggccgctcg agcagaaact catctcagaa gaggatctgg
     5401 cagcaaatga tatcctggat tacaaggatg acgacgataa ggtttaaacg gccggccgcg
     5461 gtcatagctg tttcctgaac agatcccggg tggcatccct gtgacccctc cccagtgcct
     5521 ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt
     5581 tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta
     5641 tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca
     5701 agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg
     5761 attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc
     5821 taatttttgt ttttttggta gaggcggggt ttcaccatat tggccaggct ggtctccaac
     5881 tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga
     5941 accactgctc ccttccctgt ccttctgatt ttaaaataac tataccagca ggaggacgtc
     6001 cagacacagc ataggctacc tggccatgcc caaccggtgg gacatttgag ttgcttgctt
     6061 ggcactgtcc tctcatgcgt tgggtccact cagtagatgc ctgttgaatt gggtacgcgg
     6121 ccagcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     6181 ggataacgca ggaaagaaca tgtccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6241 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6301 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6361 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6421 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6481 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6541 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6601 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6661 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6721 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6781 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6841 acggggtctg acgctcagtg gaacgacgcg taactcacgt taagggattt tggtcatgag
     6901 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6961 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     7021 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7081 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7141 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7201 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7261 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     7321 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7381 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7441 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7501 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7561 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7621 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7681 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7741 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7801 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7861 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgatga tattatttta
     7921 tcttgtgcaa tgtaacatca gagattttga gacacgggcc agagctgcca ggaaacagct
     7981 atgaccatgt aatacgactc actatagggg atatcagctg gatggcagtt aac
//
