Figure 3.
Engineering diagram for stitching of the epothilone gene cluster. pSuperCos-epo35 and pSuperCos-epo14 were the starting clones, which include an overlap in the epoD gene. pSuperCos-epo35 was retrofitted with the p15A origin and the chloramphenicol resistance gene from pACYC184 by subcloning to remove the pSuperCos backbone, introduce the short homology arm ‘(A)’ and create p15A-epo35. pSuperCos-epo14 was digested with ScaI and the epoA-D genes were recombined into p15A-epo35 by triple recombination using a bridging zeo. In (B), the NcoI digest reveals the correct product. (the NcoI sites in p15A-epo35 and p15A-epo are shown in Appendix 1 in Supplementary Material).