Significance DNA variant libraries allow unbiased analysis of sequence–function relationships. However, exhaustive combinatorial assemblies of even a modest number of parts produce libraries too large to practically screen. Library size can be reduced by manually blending parts to remove unwanted combinations, but this yields limited improvements and is difficult to scale. To address this challenge, we built a microfluidic hybrid valve–droplet device that selectively combines and assembles DNA parts into libraries of predefined composition with low bias. We use this system to construct and study a library of engineered histidine kinases, revealing how structural changes are communicated across adjacent domains to modulate kinase activity. Our method creates rationally reduced libraries and has applications in synthetic biology and protein engineering.