pSL103-SpCas9-FLAG-3XNLS
(Plasmid #182032)

• Purpose: Expression of recombinant Streptococcus pyogenes Cas9 protein carrying C-terminal FLAG and 3XNLS for genome editing as Cas9 RNP
• Depositing Lab: Steven Lin
• Publication: Lin et al Bio Protoc. 2022 May 20;12(10):e4420. doi: 10.21769/BioProtoc.4420. eCollection 2022 May 20.

Backbone

• Vector backbone: Synthetic vector
• Backbone size w/o insert (bp): 6484
• Total vector size (bp): 10762
• Vector type: Bacterial Expression, CRISPR

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: Streptococcus pyogenes cas9
• Alt name: SpCas9
• Species: Synthetic
• Insert Size (bp): 4278
• Entrez Gene: cas9 (a.k.a. SD90_RS03825, SD90_03830)
• Promoter: T7
• Tags / Fusion Proteins:
  • MBP (N terminal on backbone)
  • FLAG (C terminal on insert)
  • 3XNLS (C terminal on insert)
  • 12X His tag (C terminal on backbone)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: GACGCGCAGACTAATTCG
• 3′ sequencing primer: GCTAGTTATTGCTCAGCGG

Resource Information

• Supplemental Documents:
  • pSL103-SpCas9-FLAG-3xNLS (Addgene deposit).gb

Terms and Licenses

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

Depositor Comments

The ORF of cas9 is codon optimized for E. coli expression and was synthesized by GeneArt.
The additional features (MBP, TEV sites, FLAG, 3XNLS and 12XHis tag) were either PCR amplified from various vectors or purchased as synthetic DNA fragments. All gene fragments were assembled by Gibson cloning into this customized vector.