A method called PERMutation Using Transposase Engineering (PERMUTE) is described for creating libraries of circularly permuted proteins. This approach uses the transposase MuA to randomly insert a minitransposon into a plasmid containing the open reading frame (ORF) of interest. The minitransposon functions as a protein expression vector, and after integration, the ORF is circularized through intramolecular ligation. This method generates a library of vectors that express different permuted variants of the ORF-encoded protein. The PERMUTE approach produces vectors with distinct sequence diversity compared to existing methods. A library of Thermotoga neapolitana adenylate kinase (AK) was constructed using PERMUTE, and selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE is useful for directed evolution of proteins with new functions. The method involves using transposase to introduce a unique restriction site into the gene, allowing for random insertion of the minitransposon. This approach minimizes deletions and duplications that occur with traditional methods. The PERMUTE method was tested by applying it to AK from Thermotoga neapolitana, a thermostable enzyme. The resulting library was selected for variants that complement E. coli growth at 40°C, revealing functional proteins with novel architectures. The library was characterized, and it was found that the PERMUTE method generates a diverse set of permuted proteins with distinct sequence diversity. The method also allows for the creation of a library of circularly permuted proteins with a high degree of sequence diversity, which can be used for directed evolution and protein engineering. The PERMUTE method is useful for future studies exploring protein fitness landscapes through directed evolution and for evaluating the effect of protein thermostability on protein tolerance to permutation type mutations. It also has potential applications in the construction of molecular switches for synthetic biology. The method is efficient and avoids the deletions and duplications that occur with traditional methods, making it a valuable tool for protein engineering.A method called PERMutation Using Transposase Engineering (PERMUTE) is described for creating libraries of circularly permuted proteins. This approach uses the transposase MuA to randomly insert a minitransposon into a plasmid containing the open reading frame (ORF) of interest. The minitransposon functions as a protein expression vector, and after integration, the ORF is circularized through intramolecular ligation. This method generates a library of vectors that express different permuted variants of the ORF-encoded protein. The PERMUTE approach produces vectors with distinct sequence diversity compared to existing methods. A library of Thermotoga neapolitana adenylate kinase (AK) was constructed using PERMUTE, and selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE is useful for directed evolution of proteins with new functions. The method involves using transposase to introduce a unique restriction site into the gene, allowing for random insertion of the minitransposon. This approach minimizes deletions and duplications that occur with traditional methods. The PERMUTE method was tested by applying it to AK from Thermotoga neapolitana, a thermostable enzyme. The resulting library was selected for variants that complement E. coli growth at 40°C, revealing functional proteins with novel architectures. The library was characterized, and it was found that the PERMUTE method generates a diverse set of permuted proteins with distinct sequence diversity. The method also allows for the creation of a library of circularly permuted proteins with a high degree of sequence diversity, which can be used for directed evolution and protein engineering. The PERMUTE method is useful for future studies exploring protein fitness landscapes through directed evolution and for evaluating the effect of protein thermostability on protein tolerance to permutation type mutations. It also has potential applications in the construction of molecular switches for synthetic biology. The method is efficient and avoids the deletions and duplications that occur with traditional methods, making it a valuable tool for protein engineering.