Figure 1. Schematic diagram of antibiotic gene cluster cloning from Streptomyces chromosome.

Initially, a pUC119-based suicide plasmid (pSV::attB₆Up) carrying attB₆ and a region homologous to 5′ end of the cluster is introduced into the chromosome by a single crossover. A second plasmid pKC1139::attP₆Dn is based on pKC1139 carrying attP₆ and a region homologous to 3′ end of the cluster. When the incubation temperature is higher than 34°C, pKC1139::attP₆Dn turns into a non-replicating plasmid and then is integrated into the chromosome by a single crossover. Expression of ϕBT1 integrase (encoded in the plasmid pIJ10500) leads to excision of the pKC1139 backbone with gene cluster of interest, leaving behind the suicide vector pUC119::neo and 42 bp attL6 site. aac(3)IV: apramycin resistance gene; neo: kanamycin resistance gene; ori: temperature-sensitive origin of replication from pSG5; rep: rep encoding a replication initiator protein from pSG5.