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Construction and characterization of a new cloning shuttle vector for gene transfer and expression in Bacillus thuringiensis.A novel short and high-copy number shuttle vector called pHBLBIV, was constructed for gene transfer and expression in Bacillus thuringiensis. A 1·6-kbp replicon of a relatively high-copy number endogenous plasmid of a selected B. thuringiensis strain was ligated to Escherichia coli pUC18 replicon containing the ampicillin and the erythromycin resistance genes used for the selection of respectively E. coli and B. thuringiensis transformants. The constructed vector was shown to have a high copy number compared with the conventional B. thuringiensis vectors, and used successfully for the transfer of vegetative insecticidal protein-encoding gene (vip) in between B. thuringiensis strains.A new shuttle vector of B. thuringiensis–E. coli named pHBLBIV was constructed. It was characterized by its high copy number, small size and segregational stability. This vector was successfully used for vip gene cloning and transfer in B. thuringiensis.A novel shuttle vector has been constructed, which has demonstrated potential for the cloning and expression of genes in B. thuringiensis.