As a bacteriophage lambda insertion vector,λET-CLONE has the advantages of high cloning efficiency, stability, and ease of screening by high-density plaque lifts. cDNA up to 8 kbp in size can be inserted directionally into EcoR I/Hind III arms.The cloning region is located downstream of T7 expression signals and vector-encoded peptides so fusion proteins containing the 260-aa T7 Tag, and 6-aa His tag sequences can be produced in appropriate hosts. Libraries can thus be screened for expressed polypeptides with antibodies or other ligands, as well as with conventional nucleic acid probes. Plasmid subclones are produced by site-specific recombination at dual loxP sites in the vector; colonies containing pET-CLONE subclones are obtained simply by plating recombinants on an appropriate host in the presence of ampicillin (or carbenicillin). The plasmid moiety contains the same expression elements as pET vectors and is suitable for high-level expression of most target proteins without the need for further subcloning. The fusion tags enable the use of a variety of strategies for detection and purification of target proteins, and are removable with thrombin or enterokinase. An SP6 promoter proximal to the cloning sites also enables synthesis of non-fused RNA for probes and in vitro translation.