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The need for co-ordinate, high-level, and stable expression of multiple genes is essential for the engineering of biosynthetic circuits and metabolic pathways. This work outlines the functionality and design of IRES- and 2 A-peptide-based constructs by comparing different strategies for co-expression in polycistronic vectors. In particular, 2 A sequences are small peptides, mostly derived from viral polyproteins, that mediate a ribosome-skipping event such that several, different, separate proteins can be generated from a single open reading frame. When applied to metabolic engineering and synthetic gene circuits, 2 A peptides permit to achieve co-regulated and reliable expression of various genes in eukaryotic cells.