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Construction of CPEB mutants
Point mutations were made with a Chamaleon mutagenesis kit (Stratagene). The selection primer was located in plasmid pMycCPEB5, 5[H11032]-CCTCGAGGGGCGGGCCCGTACCCAATTCGCCC-3[H11032], and changed a KpnI site to a SrfI site. This primer was used in conjunction with the following mutation primers to create new clones: serines 174 and 180 to alanine (CPEB-AA), 5[H11032]-GCTCTCGATTGGACGCCCGGTCTATTTTGGATGCTCGCTCC-3[H11032]; and serines 174 and 180 to aspartic acid (CPEB-DD), 5[H11032]-GCTCTCGATTGGACGATCGCTCTATTTTGGATGATCGCTCC-3[H11032]. The His-tagged form of the mutant CPEBs was obtained by subcloning the NcoIBamHI fragment of CPEB-AA and CPEBDD into the NcoI and BamHI sites of pHisCPEB6.
Received 15 November 1999; accepted 24 January 2000.
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