Cis-regulatory sequences control when, where, and how much genes are transcribed. A better understanding on these elements is a fundamental keystone to better understand development, cell differentiation, and morphogenesis. Several methods based on in silico analysis or ChIP-seq experiments have been developed to detect cis-acting sequences. Here, we describe a protocol to isolate such sequences from genomic DNA and to clone them into expression vectors for functional assays using the Gateway cloning technology.