The average size of natural product biosynthetic gene clusters (10-200 kb) often makes it impractical to genetically manipulate them using traditional restriction/ligation methods. In recently years, other powerful alternative recombinogenic methods, such as PCR-targeting methods, have been developed to circumvent these problems. Here we describe a new generic technique that can be used to reconstruct large biosynthetic pathways from partial gene clusters present on separate cosmid vectors. We employed this technique to reconstruct the complete anthramycin biosynthetic gene cluster from thermotolerant actinomycete Streptomyces refuineus from two donor cosmids, each with a fragment of the anthramycin biosynthetic gene cluster, into a single cosmid vector. Subsequently, this cosmid was retrofitted with an origin of transfer, C31 integrase and recombination site and transformed into the heterologous host Streptomyces lividans TK24. Anthramycin production was observed in the recombinant host, which confirms integrity of whole anthramycin biosynthetic pathway from a single cosmid vector.