Sequence features, including the affinity of binding motifs for their cognate transcription factors, are important contributors to promoter behavior. The ability to predictably recode affinity enables the development of synthetic promoters with varying levels of response to known cellular signals. Here we describe a luminescence-based microplate assay for comparing the interactions of transcription factors with short DNA probes. We then demonstrate how these data can be used to design synthetic plant promoters of varying strengths that respond to the same transcription factor.