pET21b(+)_SARS-CoV-2_3CLpro-Q306A
(Plasmid
#177334)
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Purposebacterial expression of active SARS-CoV-2 3C-like proteinase
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 177334 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 * |
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Backbone
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Vector backbonepET21b(+)
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Backbone manufacturerNovagen
- Backbone size w/o insert (bp) 5400
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Vector typeBacterial Expression
Growth in Bacteria
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Bacterial Resistance(s)Ampicillin, 100 μg/mL
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Growth Temperature37°C
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Growth Strain(s)DH5alpha
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Growth instructionsBL21(DE3) for expression.
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Copy numberLow Copy
Gene/Insert
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Gene/Insert nameSARS-CoV-2 3C-like proteinase (ORF1ab Severe acute respiratory syndrome coronavirus 2)
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Alt namensp5A_3CLpro and nsp5B_3CLpro
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Alt namemain proteinase (Mpro)
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SpeciesSARS-CoV-2 virus
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Insert Size (bp)1074
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Mutationsilent mutation C to T at position 10546 (eliminating NdeI site); codon CAA for Q306 to codon GCA for A306 (eliminating natural 3CL autocleavage site)
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GenBank IDNC_045512.2
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Entrez GeneORF1ab (a.k.a. GU280_gp01)
- Promoter T7
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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
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Academic/Nonprofit Terms
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Industry Terms
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; Factor Xa cleavage site can cleave off all 3 downstream tags; three tandem FLAG epitope tags is followed by an enterokinase cleavage site.
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.
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For your Materials & Methods section:
pET21b(+)_SARS-CoV-2_3CLpro-Q306A was a gift from Christopher Overall (Addgene plasmid # 177334 ; http://n2t.net/addgene:177334 ; RRID:Addgene_177334) -
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