Donor Plasmid Construction for Bac-to-Bac™ Baculovirus Expression System

We used the Bac-to-Bac™ Baculovirus Expression System (Thermo Fisher Scientific) to obtain the recombinant baculovirus BacDual-3BT, which was designed to express the 3BT-GS protein on the baculoviral membrane and in transduced mammalian cells. A synthetic cassette containing the promoters P10, CMV, and PH in tandem, was synthesized by Biomatik (Kitchener, CA) and cloned into pFastBac™ Dual (Thermo Fisher Scientific) in the restriction sites XhoI and EcoRI. The reporter gene egfp was PCR-amplified from pEGFP-C1 (Clontech), ligated with pTZ57R/T (Thermo Fisher Scientific), and excised with KpnI and SphI for subcloning into pFastBac™ Dual. The chimeric intron from the human β-globin and immunoglobulin heavy chain genes was PCR-amplified from the plasmid pCI-neo (Promega Corporation), ligated with pTZ57R/T, excised with NotI and HindIII, and cloned into pFastBac™ Dual, obtaining the vector pFBD-EGFP. To create the final expression vector pFBD-3BT-GS, the synthetic 64-3bt-gs gene was cloned into pFBD-EGFP in SacI and BamHI ( Supplementary Figure 4 ). The clones were analyzed by endpoint PCR, restriction digestion, and Sanger sequencing.