We established and characterized a reporter cell line for testing how the genomic distance to a target promoter affects single enhancers and combinations thereof.

Enhancers are short DNA sequences that activate their target promoter from a distance; however, increasing the genomic distance between the enhancer and the promoter decreases expression levels. Many genes are controlled by combinations of multiple enhancers, yet the interaction and cooperation of individual enhancer elements is not well understood. Here, we developed a novel synthetic platform that allows building complex regulatory landscapes from the bottom up. We tested the system by integrating individual enhancers at different distances and confirmed that the strength of an enhancer contributes to how strongly it is affected by increased genomic distance. Furthermore, synergy between two enhancer elements depends on the distance at which the two elements are integrated: introducing a weak enhancer between a strong enhancer and the promoter strongly increases reporter gene expression, allowing enhancers to activate from increased genomic distances.