Protocol

Abbreviation

WU_baculovirus_cloning_1

Name

WU_baculovirus_cloning_1

Laboratory name

Washington University

Type

cloning

Description

Insertion of synthetic g-block DNAs encoding soluble targets into a modified baculovirus transfer vector by Gibson Assembly method. The pOET1 vector was modified to include a signal peptide from baculovirus gp64 envelope glycoprotein (MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA). Also, after the ApaI cloning site, a thrombin cleavage site, a foldon trimerization domain, and a 6-His affinity purification tag were added to be in frame with the target sequence (RSLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH). The vector was linerized with ApaI and dephosphorylated with Shrimp Alkaline Phosphatase before joining.