PX330 (2nd Gen)

BbsI Transfection  hU6

pX330-U6-Chimeric_BB-CBh-hSpCas9

Mammalian Expression, CRISPRCBhhumanized S. pyogenes Cas9

Map

• PURPOSE

  A human codon-optimized SpCas9 and chimeric guide RNA expression plasmid.

• 
  

• Vector backbone: pUC ori vector
• • Vector type
    Mammalian Expression, CRISPR

  GROWTH IN BACTERIA

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    Stbl3
  • Copy number
    High Copy

  
  

• 
  

• GENE/INSERT

  • Gene/Insert name
    humanized S. pyogenes Cas9
  • Alt name
    SpCas9
  • Alt name
    hSpCas9
  • Insert Size (bp)
    4272

  • PromoterCBh

  • Tag / Fusion Protein

    • 3xFLAG (N terminal on insert)

    
    

  CLONING INFORMATION

  • Cloning methodRestriction Enzyme

  • 5′ cloning siteAgeI (not destroyed)

  • 3′ cloning siteEcoRI (not destroyed)

  • 5′ sequencing primeragggatggttggttggtggg

  • 3′ sequencing primerCCAATCCTCCCCCTTGCTGT

  
  

• 
  

• Alternate plasmid name: pSpCas9(BB) (PX330)

  For plasmid usage, please see the associated publication (Cong et al. Science. 2013, PMID: 23287718), as well as Ran et al. Nat Protoc. 2013, PMID: 24157548.

  
  

  SEQUENCE INFORMATION

  >pX330-U6-Chimeric_BB-CBh-hSpCas9 sequence 8506 bps

  gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaa

  ttaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggta

  gtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgat

  ttcttggctttatatatcttGTGGAAAGGACGAAACACCggGTCTTCgaGAAGACctgttttagagctaG

  AAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTg

  ttttagagctagaaatagcaagttaaaataaggctagtccgtTTTTagcgcgtgcgccaattctgcagac

  aaatggctctagaggtacccgttacataacttacggtaaatggcccgcctggctgaccgcccaacgaccc

  ccgcccattgacgtcaatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacgg

  taaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacg

  gtaaatggcccgcctggcattGtgcccagtacatgaccttatgggactttcctacttggcagtacatcta

  cgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctccccc

  ccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcgggggggg

  ggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggc

  ggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctat

  aaaaagcgaagcgcgcggcgggcgggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgcc

  gcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggccctt

  ctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaat

  gtttaattacctggagcacctgcctgaaatcactttttttcaggttGGaccggtgccaccATGGACTATA

  AGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAA

  GAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATC

  GGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGC

  TGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAAC

  AGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTAT

  CTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCT

  TCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTA

  CCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTG

  CGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGA

  ACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGA

  AAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGG

  CTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGA

  GCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAA

  GGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTG

  GCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGG

  CCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCT

  CGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGC

  TACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACG

  GCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGG

  CAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCA

  TTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTC

  TGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTT

  CGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAAC

  CTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGA

  CCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCAT

  CGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAA

  ATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACC

  ACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGA

  TATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCAC

  CTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGA

  AGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTT

  CGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCC

  CAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGA

  AGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAA

  CATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATG

  AAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCC

  AGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACT

  GGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCC

  ATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGG

  TCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGA

  CAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTG

  GTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACG

  AGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAA

  GGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCC

  GTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGG

  TGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTT

  CTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCT

  CTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGA

  AAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGA

  GTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTAC

  GGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCA

  AGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCC

  CATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTAC

  TCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACG

  AACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTC

  CCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAG

  CAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACA

  ACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCT

  GGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAG

  GTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGC

  TGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGtaagaattcCT

  AGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGC

  CTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTG

  TCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAg

  AATAGCAGGCATGCTGGGGAgcggccgcaggaacccctagtgatggagttggccactccctctctgcgcg

  ctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagt

  gagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggt

  atttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgt

  ggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccct

  tcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgat

  ttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgcc

  ctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaact

  ggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctatt

  ggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttt

  atggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacaccc

  gctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccggga

  gctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcct

  atttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtg

  cgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct

  gataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcc

  cttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaa

  gatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttc

  gccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtat

  tgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcacca

  gtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtg

  ataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaa

  catgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgag

  cgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactc

  tagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggc

  ccttccggctggctggtttattgctgataaatctggagccggtgagcgtggaagccgcggtatcattgca

  gcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatgg

  atgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagt

  ttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctt

  tttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaa

  agatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccacc

  gctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagc

  agagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtag

  caccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtct

  taccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgc

  acacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcg

  ccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcac

  gagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgag

  cgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttac

  ggttcctggccttttgctggccttttgctcacatgt