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DNA-binding sites for SYCRP1, which is a regulatory protein of the cyanobacterium Synechocystis sp. PCC6803, were predicted for the whole genome sequence by estimating changes in the binding free energy (ΔΔAtotal) for SYCRP1 for those sites. The ΔΔAtotal values were calculated by summing ΔΔG values derived from systematic single base-pair substitution experiments (symmetrical and cooperative binding model). Of the calculated binding sites, 23 sites with a ΔΔAtotalvalue < 3.9 kcal·mol−1 located upstream or between the ORFs were selected as putative binding sites for SYCRP1. In order to confirm whether SYCRP1 actually binds to these binding sites or not, 11 sites with the lowest ΔΔAtotal values were tested experimentally, and we confirmed that SYCRP1 binds to ten of the 11 sites with a ΔΔGtotal value < 3.9 kcal·mol−1. The best correlation coefficient between ΔΔAtotal and the observed ΔΔGtotal for binding of SYCRP1 to those sites was 0.78. These results suggest that the ΔΔG values derived from systematic single base-pair experiments may be used to screen for potential binding sites of a regulatory protein in the genome sequence.