Lentiviral infection is often used to integrate genetic material into cells to stably express transgenes of interest. Depending on the location of integration into the host genome, readthrough expression of the lentiviral cargo can occur via an upstream endogenous promoter, which is typically an unwanted phenomenon because it can result in dysfunctional expression. The purpose of this study was to demonstrate that readthrough expression can be a wanted phenomenon for expressing functional proteins while at the same time reducing the size of the lentiviral transfer plasmid. Readthrough expression was used to generate HEK293 cell lines stably expressing fluorescent reporter proteins, reporter protein-antibiotic resistance fusion proteins for selection, and the vascular endothelial growth factor receptor 2. The generated proteins were all functional, as demonstrated by their ability to fluoresce, confer antibiotic resistance, and participate in receptor-mediated signalling, respectively. Therefore, we suggest that the mechanism of readthrough expression may have further applications in the expression of larger genes or genetic circuits (e.g. cell-based therapeutics), where the lentiviral cargo limit is stretched to the maximum.