 The study examined the effect of transcription factor binding site, TFBS, orientation and order on gene regulation. It found that TFBS orientation and order had a significant effect on gene regulation, which was corroborated by genomic analysis. Furthermore, it showed that TFBS orientation improved the accuracy of a model used to predict gene expression from sequence. This article was authored by Ilias Jorga-Copopoulos Aureus, Chinu Deng, the Kramugawal, and others.