Concentrations and volumes for synthesis of the SARS-CoV-2 pseudoknot.

	Component	Stock concentration	Volume added (μL)
PCR	Nuclease-free water	N/A	30
	Q5 reaction buffer (NEB)	10×	10
	dNTPs	10 mM	1
	Forward primer	10 mM	2.5
	Reverse primer	10 mM	2.5
	DNA template	50 ng μL−1	1
	DMSO	N/A	2.5
	Q5 polymerase (NEB)	2000 U mL−1	0.5
IVT	Nuclease-free water	N/A	18.2
	MgCl2	1 M	1
	Tris–HCl, pH 8.0	1 M	4
	rNTP mix	10 mM	3
	Spermidine	0.1 M	1
	DTT	1 M	0.1
	Pyrophosphatase (NEB)	100 U μL−1	0.2
	DNA from PCR	28–35 ng μL−1	10
	T7 polymerase (Tolbert lab)	50 000 U mL−1	2.5
	DMSO	N/A	10