To construct a cyclin-luciferase fusion protein (pSP cyc-luc), the N-terminal sequence of Xenopus laevis cyclin B1, including amino acids 2-97, was amplified by PCR, digested with BstEII, and ligated into the pSP-lucNF expression vector (Promega).The resulting vector was sequence verified. The fusion protein was expressed by coupled in vitro transcription and translation in reticulocyte lysate using the SP6-TNT Coupled Reticulocyte Lysate System (Promega) and flash frozen in liquid nitrogen until the time of use. The parental pSP-lucNF vector was used to express unmodified luciferase.A vector for expression of cyclin-luciferase in E. coli (pET cyc-luc) was also constructed; this protein behaved identically in all assays to the protein expressed in reticulocyte lysate, but could be made in higher quantities necessary for screening. pSP cyc-luc was digested with HindIII and XhoI. The resulting 1949 bp fragment containing the cyclin B1-luciferase sequence was ligated into the pET 28b expression vector
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