Identification and validation of promoters and cis-acting regulatory elements

•We present an overview of native promoter structure and function.•The use and application of promoters in transgenics are emphasized.•Use of cis-acting regulatory elements in synthetic promoters is highlighted.•A new approach of developing and using synthetic introns in promoters is presented.•Different promoter gene expression validation approaches are compared. Studies of promoters that largely regulate gene expression at the transcriptional level are crucial for improving our basic understanding of gene regulation and will expand the toolbox of available promoters for use in plant biotechnology. In this review, we present a comprehensive analysis of promoters and their underlying mechanisms in transcriptional regulation, including epigenetic marks and chromatin-based regulation. Large-scale prediction of promoter sequences and their contributing cis-acting elements has become routine due to recent advances in transcriptomic technologies and genome sequencing of several plants. However, predicted regulatory sequences may or may not be functional and demonstration of the contribution of the element to promoter activity is essential for confirmation of regulatory sequences. Synthetic promoters and introns provide useful approaches for functional validation of promoter sequences. The development and improvement of gene expression tools for rapid, efficient, predictable, and high-throughput analysis of promoter components will be critical for confirmation of the functional regulatory element sequences identified through transcriptomic and genomic analyses.