TnphoA is a transposon that enables the creation of hybrid proteins combining alkaline phosphatase (phoA) with amino-terminal sequences of other proteins. This system allows the identification of export signals in proteins, such as transmembrane proteins, and helps locate genes for secreted and transmembrane proteins. The transposon TnphoA inserts into a gene to generate fusions of alkaline phosphatase to the amino-terminal sequences of the protein product of that gene. The study shows that alkaline phosphatase can be exported when fused to sequences from secreted periplasmic proteins or complex cytoplasmic membrane proteins, even though these proteins generally lack cleaved signal sequences. TnphoA was constructed by inserting phoA sequences into IS50, followed by recombination into the complete Tn5. The DNA sequence of the left end of TnphoA was determined, revealing that it contains 50 base pairs of DNA, with 48 derived from IS50L and 2 from a Pst I linker. The amino acid residues encoded by this 50-bp sequence are present at the fusion joint of every hybrid protein generated by TnphoA insertion. The DNA sequence differs from that of IS50L by an A-to-G change at position 29, which eliminates the opal nonsense codon in frame with the alkaline phosphatase coding sequence. The study also shows that the stability and localization of hybrid proteins depend on the presence of functional signal sequences. Hybrid proteins with functional β-lactamase signal sequences were exported to the periplasm and showed alkaline phosphatase activity. In contrast, hybrid proteins lacking these sequences were not exported and showed no activity. The stability of hybrid proteins was examined using pulse-chase experiments, revealing that longer hybrid proteins were more stable. The locations of different bla-phoA hybrid proteins were determined by cell fractionation, showing that shorter hybrid proteins fractionated to the periplasm, while longer ones were found in the membrane fraction. The study also found that fusions to cytoplasmic proteins like chloramphenicol transacetylase and β-galactosidase resulted in very low or undetectable enzyme activity. The use of TnphoA combines the advantages of working with hybrid proteins that can be secreted with the versatility of Tn5 transposition in generating hybrids. This system can be used to identify new genes encoding transmembrane and periplasmic proteins by their ability to generate hybrid proteins with alkaline phosphatase activity. The results suggest that TnphoA can function in a number of bacteria other than E. coli, as its parent transposon Tn5 has a broad host range for transposition.TnphoA is a transposon that enables the creation of hybrid proteins combining alkaline phosphatase (phoA) with amino-terminal sequences of other proteins. This system allows the identification of export signals in proteins, such as transmembrane proteins, and helps locate genes for secreted and transmembrane proteins. The transposon TnphoA inserts into a gene to generate fusions of alkaline phosphatase to the amino-terminal sequences of the protein product of that gene. The study shows that alkaline phosphatase can be exported when fused to sequences from secreted periplasmic proteins or complex cytoplasmic membrane proteins, even though these proteins generally lack cleaved signal sequences. TnphoA was constructed by inserting phoA sequences into IS50, followed by recombination into the complete Tn5. The DNA sequence of the left end of TnphoA was determined, revealing that it contains 50 base pairs of DNA, with 48 derived from IS50L and 2 from a Pst I linker. The amino acid residues encoded by this 50-bp sequence are present at the fusion joint of every hybrid protein generated by TnphoA insertion. The DNA sequence differs from that of IS50L by an A-to-G change at position 29, which eliminates the opal nonsense codon in frame with the alkaline phosphatase coding sequence. The study also shows that the stability and localization of hybrid proteins depend on the presence of functional signal sequences. Hybrid proteins with functional β-lactamase signal sequences were exported to the periplasm and showed alkaline phosphatase activity. In contrast, hybrid proteins lacking these sequences were not exported and showed no activity. The stability of hybrid proteins was examined using pulse-chase experiments, revealing that longer hybrid proteins were more stable. The locations of different bla-phoA hybrid proteins were determined by cell fractionation, showing that shorter hybrid proteins fractionated to the periplasm, while longer ones were found in the membrane fraction. The study also found that fusions to cytoplasmic proteins like chloramphenicol transacetylase and β-galactosidase resulted in very low or undetectable enzyme activity. The use of TnphoA combines the advantages of working with hybrid proteins that can be secreted with the versatility of Tn5 transposition in generating hybrids. This system can be used to identify new genes encoding transmembrane and periplasmic proteins by their ability to generate hybrid proteins with alkaline phosphatase activity. The results suggest that TnphoA can function in a number of bacteria other than E. coli, as its parent transposon Tn5 has a broad host range for transposition.