Application of yeast-based TAR cloning to generate viral cDNA clones and the recovery of recombinant MHV-GFP.a, Schematic representation of the general workflow of TAR cloning and virus rescue. In-yeast assembly require the delivery of overlapping DNA fragments covering the viral genome and a TAR vector into yeast in one transformation event. Transformed DNA fragments are joined by homologous recombination in yeast generating a YAC-cloned full-length viral genomic cDNA. The in vitro production of infectious capped viral RNA starts with the isolation of the YAC, followed by plasmid linearization to provide DNA template for run-off T7 polymerase-based transcription. Virus rescue starts with electroporation of BHK-21 and/or BHK-21 cells expressing the corresponding coronavirus N protein, followed by co-cultivation of electroporated cells with susceptible cell line for virus production and amplification. 