BACKGROUND: Molecular and synthetic biology tools enable the design of new-to-nature biological systems, including genetically engineered microorganisms, recombinant proteins, and novel metabolic pathways. These tools simplify the development of more efficient, manageable, and tailored solutions for specific applications, biocatalysts, or biosensors that are devoid of undesirable characteristics. The key aspect of preparing these biological systems is the availability of appropriate strategies for designing novel genetic circuits. However, there remains a pressing need to explore independent and controllable systems for the co-expression of multiple genes. RESULTS: In this study, we present the characterisation of a set of bacterial plasmids dedicated to recombinant expression in broadly used Escherichia coli. The set includes plasmids with four different, most commonly used bacterial expression cassettes - RhaS/RhaBAD, LacI/Trc, AraC/AraBAD, and XylS/Pm, which can be used alone or freely combined in up to three-gene monocistronic expression systems using Golden Standard Molecular Cloning kit assembly. The independent induction of each of the designed cassettes enables the autonomous expression of up to three recombinant proteins from one plasmid. The expression of a triple-enzyme cascade consisting of sucrose synthase, UDP-rhamnose synthase and flavonol-7-O-rhamnosyltransferase, confirmed that the designed system can be applied for the complex biocatalysts production. CONCLUSIONS: Presented herein strategy for the multigene expression is a valuable addition to the current landscape of different co-expression approaches. The thorough characterisation of each expression cassette indicated their strengths and potential limitations, which will be useful for subsequent investigations in the field. The defined cross-talks brought a better understanding of the metabolic mechanisms that may affect the heterologous expression in the bacterial hosts.