pET28a/Cas9-Cys
(Plasmid #53261)

• Purpose: Expresses N-terminal His tag fused human codon-optimized Cas9 nuclease having C-terminal Cysteine
• Depositing Lab: Hyongbum Kim
• Publication: Ramakrishna et al Genome Res. 2014 Apr 2.

Backbone

• Vector backbone: pET28a
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 5369
• Total vector size (bp): 9542
• Vector type: Bacterial Expression

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: Cas9-Cys
• Species: S. pyogenes
• Insert Size (bp): 4173
• Mutation: Additional Cysteine is added to the C-terminal region of Cas9
• Promoter: T7 Promoter
• Tags / Fusion Proteins:
  • 6X His tag (N terminal on backbone)
  • HA epitope (C terminal on insert)
  • NLS (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: Not I (not destroyed)
• 3′ cloning site: Not I (not destroyed)
• 5′ sequencing primer: TAA TAC GAC TCA CTA TAG GG
• 3′ sequencing primer: GCT AGT TAT TGC TCA GCG

Resource Information

• Articles Citing this Plasmid:
  • 18 References

Terms and Licenses

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

Depositor Comments

A sequence encoding Cas9 with a cysteine at the C-terminus was prepared by PCR
amplification using a previously described Cas9 plasmid (Cho et al, Nat Biotechnol. 2013)(Addgene: 43945) as the template and then cloned into the pET28-(a) vector (Novagen, Merk Millipore, Germany), which
includes a sequence encoding a His-tag at the N-terminus.