The constructs of native RBD and ACE2 are the same as in Zhou et al. (2020). To clone RBD N501Y, a construct of native RBD was used as the template and two primers of RBD (Forward primer 5′-CTACGGCTTTCAGCCCACATACGGTGTGGGCTACCAGCCTT-3′ and reverse primer 5′-AAGGCTGGTAGCCCACACCGTATGTGGGCTGAAAGCCGTAG-3′) and two primers of pNEO vector (Forward primer 5′- CAGCTCCTGGGCAACGTGCT-3′ and reverse primer 5′- CGTAAAAGGAGCAACATAG-3′) were used to do PCR. Amplified DNA fragments were digested with restriction enzymes AgeI and KpnI and then ligated with digested pNEO vector. This construct encodes exactly the same protein as native RBD except the N501Y mutation, as confirmed by sequencing.

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