Engineering of orthogonal systems functional across diverse hosts can benefit from employing universal regulatory DNA elements. Here, we screened a number of composite promoters in plant, fungal, and bacterial hosts, identifying variants that drive strong constitutive expression in , , , and , or only in the eukaryotic subset of these organisms. These promoters can be used in universal vectors to co-optimize for different hosts in directed evolution, engineering of biosynthetic pathways, or other biotechnological tasks that require host switching.