The effects of amino acid substitutions in the E4 sequence. Amino acid substitutions in mutants are marked in capital letters. All sequence variants were tested as β-galactosidase fusions. The expression of β-galactosidase marker protein was measured 3 h after induction with IPTG in the absence and presence of the corresponding antibiotic. The activity of each construct is shown as a ratio of Miller units measured in the absence and presence of antibiotic.