A new class of plasmid cloning vectors has been constructed with cleavage sites in a variety of translational reading phases of the promotorless lacZ gene. Fused hybrid proteins can be produced by these vectors by cloning DNA fragments containing the promoter, translation initiation site, and the amino terminal portion of a gene, all with proper orientation, into the correct translational reading frame of the lacZ gene. Enzymatically active hybrid-beta-galactosidase proteins are formed, which have amino-terminal amino acids encoded by the cloned gene segment. Another class of these vectors retains an active lac promoter and lacZ translation-initiation region, which can direct hybrid protein synthesis from DNA fragments that do not have gene initiation regions. These vectors allow transcription from the lacZ initiation region to proceed across, or to stop and restart within, an inserted fragment into the essential part of the beta-galactosidase gene. Also described is a small lacZ gene fragment (cartridge), without a plasmid replicon and without any other lac genes, which can be inserted directly into other genes to form hybrid protein fusions. Polyrestriction site sequences were easily moved into some of these vectors by incorporating drug-resistance genes that serve as markers for the selection and detection of these sequences; those markers can be easily removed afterwards.