Cloning and Mutagenesis of cGAS and 3C Protease

The N-terminal Myc/C-terminal Hemagglutinin (HA) double-tagged cGAS was ligated to pcDNA3.1 vector at the sites of HindIII and EcoRI in frame. The N-terminal FLAG/C-terminal HA double-tagged cGAS was ligated to His₆-SUMO-pET28a vector at the sites of BamHI and HindIII. N-terminal FLAG tagged 3C proteases of picornaviruses were ligated to pcDNA3.1 or 3×FLAG-CMV-10 vector. The 3C protease of SVV was ligated to His₆-SUMO-pET28a vector. Mutant plasmids of cGAS and SVV 3C protease were constructed according to the manufacturer’s instructions of Mut Express II Fast Mutagenesis Kit V2 (Vazyme, #C1002).

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Yan Y., Wu L., Yuan Y., Wang H., Yin H., Li M., Chai L., Liang R., Liu Y., Zhao D., Xing J., Li P, & Li X. (2023). Species-specific cleavage of cGAS by picornavirus protease 3C disrupts mitochondria DNA-mediated immune sensing. PLOS Pathogens, 19(9), e1011641.

Publication 2023

Mut Express II Fast Mutagenesis Kit V2

3c proteases Cgas Ecori Frame Hemagglutinin Mutagenesis Picornaviruses Plasmids Protease Vector

Corresponding Organization : Texas A&M University

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Variable analysis

independent variables

N-terminal Myc/C-terminal Hemagglutinin (HA) double-tagged cGAS
N-terminal FLAG/C-terminal HA double-tagged cGAS
N-terminal FLAG tagged 3C proteases of picornaviruses
3C protease of SVV
Mutant plasmids of cGAS and SVV 3C protease

dependent variables

No dependent variables explicitly mentioned

control variables

PcDNA3.1 vector
His6-SUMO-pET28a vector
3×FLAG-CMV-10 vector

controls

Positive control: None mentioned
Negative control: None mentioned

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