CPER reaction

SARS-CoV-2 recombinants were generated by CPER as described previously (^{Setoh et al., 2017}), with some modifications. To amplify all the cDNA fragments having complementary ends with a 25- to 452-nucleotide overlap for CPER, plasmids encoding SARS-CoV-2 gene fragments (G1–G10) and UTR linker were used as templates. The specific primers used to amplify DNA fragments (F1–F10 and the UTR linker) are described in the Key Resources Table (CoV-2-F1-Fw to CoV-2-Linker-Rv). Then, the DNA fragments of F9 and F10 were connected before CPER by overlap PCR with a primer set (CoV-2-F9-Fw and CoV-2-F10-Rv). By using equimolar amounts (0.1 pmol each) of the resulting 10 DNA fragments (F1 to F8, F9/10 and the UTR linker) and 2 μL of PrimeStar GXL DNA polymerase, CPER was performed within 50 μL reaction volumes. The cycling conditions of CPER were as follows: condition 1 (an initial 2 minutes of denaturation at 98°C; 20 cycles of 10 s at 98°C, 15 s at 55°C, and 25 minutes at 68°C; and a final extension for 25 minutes at 68°C), condition 2 (an initial 2 minutes of denaturation at 98°C; 35 cycles of 10 s at 98°C and 15 minutes at 68°C; and a final extension for 15 minutes at 68°C), or condition 3 (an initial 2 minutes of denaturation at 98°C; 35 cycles of 10 s at 98°C, 15 s at 55°C, and 15 minutes at 68°C; and a final extension for 15 minutes at 68°C).

The infectious clones of SARS-CoV-2 carrying sfGFP were also assembled by CPER using pCSII-CoV-2-G10-sfGFP as templates to acquire DNA fragment F10. The HiBiT recombinant SARS-CoV-2 was also generated by CPER. The HiBiT sequence (VSGWRLFKKIS) and a linker sequence (GSSG) were inserted in the N terminus of the ORF6 sequence by overlap PCR using DNA fragment F9 and a specific overlap primer set (ORF6-HiBiT-Rv and ORF6-HiBiT-Fw) and fragment F9-amplifying primer set (CoV-2-F9-Fw and CoV-F9-Rv). Thereafter, CPER was conducted using the resulting fragment F9 containing the HiBiT gene.