Cloning Lentiviral Vectors for SARS-CoV-2 Research

For the cloning of pLV-EIB-N and pLV-EIB-ACE2, the pLV-EF1a-IRES-Blast vector (#85133, Addgene, Watertown, MA, USA) was used. For the cloning of pLV-EIP-T7RNAP, we used the pLV-EF1a-IRES-Puro vector (#85132, Addgene). The viral nucleoprotein (N) was amplified from the patient material, T7 RNA polymerase (T7RNAP) was amplified from pCAGT7 (kindly provided by Marco Thomas, Institute for Clinical and Molecular Virology, Erlangen, Germany), and pCG1-hACE2 [25 (link)] served as the template for the amplification of hACE2. All the vectors were generated by Gibson assembly [24 (link)] and confirmed by Sanger sequencing and a Western blot analysis. For cloning pLV-EF1a-IRES-Blast-N-3xFLAG, the pBSCoV2-N-3xFLAG bacmid generated within this study was used as the template. All the primers used for plasmid generation are listed in Table S1 and were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA).