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Motivation: 'Phylogenetic footprinting' is a widely applied approach to identify regulatory regions and potential transcription factor binding sites (TFBSs) using alignments of non-coding orthologous regions from two or more organisms. A systematic evaluation of its validity and usability based on known TFBSs is needed to use phylogenetic footprinting most effectively in the identification of unknown TFBSs. Results: In this paper we use 2678 human, mouse and rat TFBSs from the TRANSFAC Òdoi:10.1093/bioinformatics/bti819 pmid:16332706 fatcat:ymuzqsd5azb37megq64sqoeckm