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Previous studies of PTKs have mapped their average preferences for amino acids surrounding phosphorylation sites, but their sensitivity to sequence variation remains poorly understood. Here, we used microbial biosensors for PTK activity to examine the influence of local sequence context on substrate specificity. Across five well-studied PTKs, we identified amino acid substitutions within consensus substrates that could confer sensitivity to substrate length or enhance selectivity for one PTK over others. Using a secondary decoy screen, we found sequence-diverse substrates with unexpectedly orthogonal PTK compatibilities. 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