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Construction of pJFW070. (A) Primers GL158 and WN008 were used to amplify the PEP synthase promoter (Ppep). Primers WN009 and WN010 were used to amplify trpAB from wild-type genomic DNA. (B) These fragments were joined by SOE PCR to produce the Ppep-trpAB marker cassette, which was treated with T4 polynucleotide kinase (PNK) and ligated into the pJFW018 fragment produced by EcoRV digestion and shrimp alkaline phosphatase (SAP) treatment, producing pJFW070.
