Figure 1.

Construction of the pNWPH-GCSF expression plasmid using prolonged overlap extension PCR/multimeric cloning strategy. Simple PCR generated 3′ and 5′ overhangs of insert (GCSF) and vector (pNWPH). These overhangs acted as primers during the formation of multimers. Circular plasmid pNWPH-GCSF was thereafter generated in B. subtilis by direct transformation of multimers containing GCSF gene. repB, replication protein B; Cat, chloramphenicol transferase gene; P_HbaII, promoter; SDgsiB, Shine-Dalgarno sequence of the gsiB gene; SPywmC, signal sequence.