pET28a(+)-SpdCas9-SBiT_Strep-Tag-II
(Plasmid #198750)

• Purpose: Expresses S. Pyogenes dCas9 fused to SmallBiT fragment of split NanoLuc luciferase
• Depositing Lab: Maarten Merkx
• Publication: van der Veer et al ACS Cent Sci. 2023 Mar 15;9(4):657-667. doi: 10.1021/acscentsci.2c01467. eCollection 2023 Apr 26.

Backbone

• Vector backbone: pET-28a(+)
• Backbone manufacturer: EMD Millipore
• Backbone size w/o insert (bp): 5369
• Total vector size (bp): 9991
• Modifications to backbone: Removed N-terminal His-tag
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: SpdCas9 - SmallBiT
• Alt name: Catalytically dead S. pyogenes Cas9 fused to SmallBiT (NanoBiT / split NanoLuc)
• Species: Synthetic; Streptococcus pyogenes
• Insert Size (bp): 4758
• Mutation: D10A, H840A (nuclease inactivating mutations in 'd' variant of SpCas9)
• Promoter: T7
• Tag / Fusion Protein:
  • Strep-tag II (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NcoI (not destroyed)
• 3′ cloning site: XhoI (not destroyed)
• 5′ sequencing primer: T7 promoter
• 3′ sequencing primer: T7 terminator

Resource Information

• Supplemental Documents:
  • pET28a(+)-SpdCas9-SBiT.gb
• A portion of this plasmid was derived from a plasmid made by: S. pyogenes dCas9 coding sequence was copied from the pET-dCas9-VP64-6xHis plasmid gifted by David Liu (Addgene plasmid #62935)

Terms and Licenses

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

Depositor Comments

This plasmid also contains the coding sequence for the LargeBiT fragment of the NanoBiT split NanoLuc luciferase downstream of the SmallBiT coding sequence, but there is a stopcodon after the strep-tag fused to SmallBiT. Hence, this plasmid encodes for the SpdCas9-SmallBiT protein by default. By cleaving the plasmid with SpeI restriction enzyme, the SmallBiT part may be removed, and after selfligation the plasmid then encodes for the SpCas9-LargeBiT protein (i.e. plasmid #198751 is obtained).