Synthetic riboswitches are attracting increasing interest for a diverse range of applications, including synthetic biology, functional genomics, and prospective therapeutic strategies. This study demonstrates that controlling alternative splicing with synthetic riboswitches represents a promising approach to effectively regulating transgene expression in mammalian cells. However, the function of synthetic riboswitches in the eukaryotic system in controlling gene expression is often limited to certain genes or cell types. So far, strategies to increase the dynamic range of regulation have been focused on adapting and modifying the riboswitch sequence itself without taking into account the context in which the riboswitch was inserted. In the present study, the tetracycline riboswitch was chosen to investigate the effects of the context and insertion site of a cassette exon within the gene to control the expression of an artificial arachidonate 5-lipoxygenase gene (