pGTM_COV2_NSP5_C145A_001_SUMO
(Plasmid #192349)

• Purpose: Expresses SARS-CoV-2 3CLpro with an active site mutation (C154A) for the expression of the mature protease in an inactive form
• Depositing Lab: Gaetano Montelione
• Publication: Mazzei et al STAR Protoc. 2023 May 5;4(2):102326. doi: 10.1016/j.xpro.2023.102326.

Backbone

• Vector backbone: pET15Sumo2_NESG
• Backbone manufacturer: Novagen/Northeast Structural Genomics Consortium
• Backbone size w/o insert (bp): 5737
• Total vector size (bp): 6604
• Modifications to backbone: An N-terminal 6X-His tag fused to the Smt3 gene (Sumo) of Saccharomyces cerevisiae as a purification/solubility enhancement cassette. BsaI sites are included in the cloning polylinker for scarless cloning of the SARS-CoV2 3CLpro and the production of a native, mature protein after cleavage from the Sumo fusion by Sumo protease (Ulp1)
• Vector type: Bacterial Expression

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: SARS-COV-2 Mpro-C145A
• Species: SARS-CoV-2
• Insert Size (bp): 918
• Mutation: Cysteine 145 of the active site is substituted with Alanine
• GenBank ID: PRO_0000449623
• Entrez Gene: NEWENTRY
• Promoter: T7 lac
• Tag / Fusion Protein:
  • 6X-His-Sumo (N terminal on backbone)

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: T7 Forward
• 3′ sequencing primer: T7 Reverse

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Genscript was contracted to produce this construct

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Industry MTA