Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoters systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron / exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptors (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual CAR cassettes, is limited.