Four different permissive sites in ANT were evaluated with six Im7 variants spanning a wide range of in vitro stabilities. The MIC values were determined for the following strains, from left to right: for the insertion site at amino acid (AA) 153, strains AMS18, AMS19, AMS17, AMS16, AMS20, and AMS21; for the insertion site at amino acid 155, AMS6, AMS4, AMS8, AMS2, AMS7, and AMS9; for the insertion site at amino acid 157, AMS24, AMS25, AMS23, AMS22, AMS26, and AMS27; and for the insertion site at amino acid 289, AMS30, AMS31, AMS29, AMS28, AMS32, and AMS33. Independently of the exact location of the permissive site in ANT, we found that all biosensors showed a clear correlation between the in vitro stabilities of the Im7 inserts and the antibiotic resistance of the corresponding fusions.