Building and using sequence‐expression‐activity maps (SEAMAPs) AThe RBS Library Calculator designs a synthetic RBS library to efficiently search a multi‐dimensional protein expression space. Building and using sequence‐expression‐activity maps (SEAMAPs) AThe RBS Library Calculator designs a synthetic RBS library to efficiently search a multi‐dimensional protein expression space. A kinetic mechanistic model maps the relationship between protein expression levels and genetic system activity, using a minimal number of measurements for parameterization. The SEAMAP's predictions are repeatedly used for different design objectives. The RBS Library Calculator designs a new RBS library to zoom onto a region of targeted protein expression levels for optimized genetic system performance. BThe RBS Library Calculator combines a biophysical model of translation with a genetic algorithm to identify the smallest degenerate RBS sequence (dRBS) with maximal search coverage for an input protein‐coding sequence (CDS). The biophysical model calculates the ribosome's binding free energy ΔGtot for an input mRNA sequence S, which is then related to its translation initiation rate and protein expression level P. C–EFluorescence measurements show that optimized RBS libraries in Escherichia coli DH10B searched a 1‐dimensional expression level space with 94, 79, and 99% search coverages at high, medium, and low search resolutions, respectively. Translation initiation rate predictions (red diamonds) are compared to measurements (Pearson R2 is 0.88, 0.79, and 0.89, respectively. All P‐values < 0.001). Data averages and standard deviations from 6 measurements. Iman Farasat et al. Mol Syst Biol 2014;10:731 © as stated in the article, figure or figure legend