pET21b(+)_SARS-CoV-2_3CLpro-Q306A
(Plasmid #177334)

• Purpose: bacterial expression of active SARS-CoV-2 3C-like proteinase
• Depositing Lab: Christopher Overall
• Publication: Pablos et al Cell Rep. 2021 Oct 26;37(4):109892. doi: 10.1016/j.celrep.2021.109892. Epub 2021 Oct 9.

Backbone

• Vector backbone: pET21b(+)
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 5400
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: BL21(DE3) for expression.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: SARS-CoV-2 3C-like proteinase (ORF1ab Severe acute respiratory syndrome coronavirus 2)
• Alt name: nsp5A_3CLpro and nsp5B_3CLpro
• Alt name: main proteinase (Mpro)
• Species: SARS-CoV-2 virus
• Insert Size (bp): 1074
• Mutation: silent mutation C to T at position 10546 (eliminating NdeI site); codon CAA for Q306 to codon GCA for A306 (eliminating natural 3CL autocleavage site)
• GenBank ID: NC_045512.2
• Entrez Gene: ORF1ab (a.k.a. GU280_gp01)
• Promoter: T7
• Tag / Fusion Protein:
  • Factor Xa site-3xFlag-Myc-6xHis (C terminal on insert)

Cloning Information

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

Terms and Licenses

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

Depositor Comments

Silent mutation in the original sequence to delete a second NdeI site as NdeI was needed for cloning; codon coding for Q306 was mutated to codon coding for A306 to delete natural cleavage site of the 3CL-protease; Factor Xa cleavage site can cleave off all 3 downstream tags; three tandem FLAG epitope tags is followed by an enterokinase cleavage site.