pGFP-PS1-sgRNA1-reporter-SpCas9_pSC101-RepA-N99D
(Plasmid #262852)

• Purpose: GFP reporter carrying protospacer PS1 and a constitutive SpCas9 sgRNA cassette, for the in vivo nicking screen in E. coli. Kanamycin, pSC101 RepA N99D.
• Depositing Lab: John van der Oost
• Depositing Organization: Wageningen University, Department Agrotechnology & Food Sciences
  Located in the Netherlands
• Publication: Size and sequence variation of the Lid of the Cas12a nuclease domain results in a nickase phenotype (unpublished)

Backbone

• Vector backbone: pSC101 (RepA N99D)
• Backbone size w/o insert (bp): 3720
• Total vector size (bp): 4437
• Modifications to backbone: Protospacer PS1 inserted by KpnI/AatII ligation of annealed oligos BG24541/BG24542. SpCas9 sgRNA cassette (J23119 promoter, spacer 1, gRNA scaffold) introduced at the AfeI/AvrII sites with BG24545/BG24546. The target sites lie in non-coding sequence, not in the GFP gene.
• Vector type: Bacterial Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Cloned and propagated in E. coli DH10B, LB at 37 C with kanamycin. Used together with a pCas SpCas9 plasmid (chloramphenicol) in the in vivo nicking screen; loss of this reporter reports cleavage.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: GFP
• Alt name: gfp
• Species: Synthetic
• Insert Size (bp): 717
• Promoter: J23119 (sgRNA cassette)

Cloning Information

• Cloning method: Golden Gate
• 5′ sequencing primer: TACCAGAGGGCGCCCCAG
• 3′ sequencing primer: CATGCAAGCTCTAGAGG

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry