pNS38-SadCas9-SNAP
(Plasmid #113718)

• Purpose: Bacterial vector for expression of Snap-tagged Staphylococcus aureus dCas9
• Depositing Lab: Gerald Schwank
• Publication: Savic et al Elife. 2018 May 29;7. pii: 33761. doi: 10.7554/eLife.33761.

Backbone

• Vector backbone: pET His6 MBP N10 TEV LIC cloning vector (2C-T)
• Backbone manufacturer: Scott Gradia
• Backbone size w/o insert (bp): 5938
• Total vector size (bp): 9758
• Vector type: Bacterial Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: Cas9
• Alt name: Csn1
• Species: Staphylococcus aureus
• Insert Size (bp): 3820
• Mutation: D10A, N580A
• GenBank ID: CCK74173.1
• Tags / Fusion Proteins:
  • His6-MBP-TEV (N terminal on backbone)
  • HA-2xNLS-SNAP-NLS (C terminal on insert)

Resource Information

• Supplemental Documents:
  • pNS38-SadCas9-SNAP.gbk

Terms and Licenses

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

Depositor Comments

Cloning of pNS38-SadCas9-SNAP: pAD-SaCas9-GFP was generated by replacing the SpCas9 coding sequence in pMJ922 with SaCas9 sequence using Gibson cloning (Keppler et al., 2003). QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies) was used to remove the stop codon and to introduce the D10A and N580A mutations into the SaCas9 (SadCas9) gene. Subsequently, GFP was cut out using BamHI and KpnI, and replaced by a SNAP-tag-NLS gBlock (Integrated DNA Technologies) using In-Fusion HD Cloning Plus CE (Takara).