pET28b-SpRY-His
(Plasmid #179320)

• Purpose: Plasmid for bacterial expression and purification of SpRY-Cas9
• Depositing Labs: Julián Cerón, Miguel Angel Moreno-Mateos
• Publication: Vicencio et al Nat Commun. 2022 May 12;13(1):2601. doi: 10.1038/s41467-022-30228-4.

Backbone

• Vector backbone: pET-28b-Cas9-His (#47327)
• Backbone manufacturer: Alex Schier
• Backbone size w/o insert (bp): 5271
• Total vector size (bp): 9375
• 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: For expression and purification of SpRY-Cas9, transform into Rosetta strain.
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Human codon-optimized Cas9 variant SpRY
• Alt name: SpRY
• Species: Synthetic
• Insert Size (bp): 4104
• Mutation: SpRY=A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/ N1317R/A1322R/R1333P/R1335Q/T1337R
• Tags / Fusion Proteins:
  • SV40-NLS (C terminal on insert)
  • 6xHis (C terminal on insert)

Resource Information

• Supplemental Documents:
  • pET28b-SpRY-His.gb
• A portion of this plasmid was derived from a plasmid made by: gBlock containing the indicated mutations was cloned into pET-28b-Cas9-His (#47327) linearized by PCR using primers Lin_Cas9_pET28b_fwd (5'-CTACCAAGGAGGTCCTGGACGCC-3') and Lin_Cas9_pET28b_rev (5'-GATACTTTCCTTGGAGCCTCCGG-3').

Terms and Licenses

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

Depositor Comments

This plasmid is owned by Moreno-Mateos Lab (CABD, Seville, Spain) and Cerón Lab (IDIBELL, Barcelona, Spain).

This plasmid was used to express a His-tagged SpRY-Cas9 protein in E. coli and purify it by nickel-based affinity purification.

Please visit https://www.biorxiv.org/content/10.1101/2021.06.06.447255v2 for bioRxiv preprint.