The paper describes the construction and characterization of TnphoA, a derivative of the transposon Tn5 that allows the generation of hybrid proteins composed of alkaline phosphatase (EC 3.1.3.1) lacking its signal peptide fused to amino-terminal sequences of other proteins. This hybrid protein exhibits alkaline phosphatase activity if the fused protein contributes sequences that promote export, compensating for the missing signal peptide. The study demonstrates that TnphoA can insert into genes to generate fusions with periplasmic and cytoplasmic membrane proteins, leading to enzyme activity. The results support the hypothesis that alkaline phosphatase is not active unless it is exported from the cytoplasm, and the fusion of a signal peptide can compensate for the missing signal peptide to promote export. The authors also discuss the implications of these findings for identifying export signals in complex transmembrane proteins and the potential applications of TnphoA in studying protein export mechanisms.The paper describes the construction and characterization of TnphoA, a derivative of the transposon Tn5 that allows the generation of hybrid proteins composed of alkaline phosphatase (EC 3.1.3.1) lacking its signal peptide fused to amino-terminal sequences of other proteins. This hybrid protein exhibits alkaline phosphatase activity if the fused protein contributes sequences that promote export, compensating for the missing signal peptide. The study demonstrates that TnphoA can insert into genes to generate fusions with periplasmic and cytoplasmic membrane proteins, leading to enzyme activity. The results support the hypothesis that alkaline phosphatase is not active unless it is exported from the cytoplasm, and the fusion of a signal peptide can compensate for the missing signal peptide to promote export. The authors also discuss the implications of these findings for identifying export signals in complex transmembrane proteins and the potential applications of TnphoA in studying protein export mechanisms.