Plenti cmv puro lentiviral vector

The SARS-CoV-2 N gene was PCR amplified using a 2019-nCoV_N_Positive Control (IDT 10006625) as the template, and then cloned into the pLenti-CMV-puro lentiviral vector (Addgene 17452) by Gibson assembly (NEB E1602L) (Supplementary Fig. 4a). Candidate expression vector subclones expected to carry the N gene target region were validated for full-length sequence identity by Sanger sequencing. 293F (Gibco R79007) cells (1 ×10⁶) suspended in 2 mL were co-transfected with 2 μg of pLenti-CMV-puro expression vector with or without the N-gene subclone and 1.5 μg of the psPAX2 (Addgene 12259) and 1 μg of the pMD2.G (Addgene 12259) vectors. After 48 hours, 1 mL of conditioned culture media containing lentivirus from the transfected 293F cells was added to culture wells containing 0.5×10⁶ 293F cells for 12 hours, after which cells were cultured with 1 μg/mL puromycin (Gibco A1113803) for 48 hours to select for transduced cells, which were collected and expanded in DMEM with 10% FBS to achieve cell cultures containing 3 × 10⁸ cells for EV isolation, as described above.