Objectives To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose. Results The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the P-R promoter and kanamycin resistance gene (P-R-kan(R)) at 30 degrees C, but have no effect on P-R-kan(R) gene at 37 degrees C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of similar to 10(7) cfu per mu g of genomic DNA, with no empty vectors detected. Conclusions Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.