Skip to main content
Addgene

pET21b(+)_SARS-CoV-2_3CLpro-C145A-Q306A
(Plasmid #177335)

Print

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 177335 Standard format: Plasmid sent in bacteria as agar stab 1 $85 *
Add to Cart

* Log in to view industry pricing.

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 (C145A) 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 TGT for C145 to codon GCG for A145 (eliminating active site amino acid Cysteine); 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
    • (Common Sequencing Primers)

Terms and Licenses

Trademarks:
  • Zeocin® is an InvivoGen trademark.

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; codon coding for active site residue C145 was mutated to A145 for inactive protease production; Factor Xa cleavage site can cleave off all 3 downstream tags; three tandem FLAG epitope tags is followed by an enterokinase cleavage site.

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pET21b(+)_SARS-CoV-2_3CLpro-C145A-Q306A was a gift from Christopher Overall (Addgene plasmid # 177335 ; http://n2t.net/addgene:177335 ; RRID:Addgene_177335)

  • For your References section:

    Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL(pro) substrate degradome. Pablos I, Machado Y, de Jesus HCR, Mohamud Y, Kappelhoff R, Lindskog C, Vlok M, Bell PA, Butler GS, Grin PM, Cao QT, Nguyen JP, Solis N, Abbina S, Rut W, Vederas JC, Szekely L, Szakos A, Drag M, Kizhakkedathu JN, Mossman K, Hirota JA, Jan E, Luo H, Banerjee A, Overall CM. Cell Rep. 2021 Oct 26;37(4):109892. doi: 10.1016/j.celrep.2021.109892. Epub 2021 Oct 9. 10.1016/j.celrep.2021.109892 PubMed 34672947
Related items:
Commonly requested with: