rGBD

GFP-rGBD was Addgene plasmid #26732, deposited by William Bement. The reduced expression GFP β-actin plasmid was Addgene plasmid #31502, deposited by Rick Horwitz and Tim Mitchison. In this plasmid the base pairs 91–544 of the enhancer region in the CMV promoter are deleted; herein, this promoter is called CMVdel. The rGBD was excised with BsrGI and XbaI and cloned into a demethylated and likewise digested mCherry-C1 vector (Van Unen et al., 2015). mCherry was replaced with mNeonGreen/3xmNeonGreen using the AgeI and BsrGI restriction sites. The insert mNeonGreen-rGBD and the backbone reduced expression GFP β-actin plasmid were digested with AgeI and MluI. The digested products were ligated to create CMVdel-mNeonGreen-1xrGBD. A tandem and a triple rGBD were created by PCR amplification of rGBD from CMVdel-mNeonGreen with primers shown in Table S1 and digestion with BsrGI and SalI. The backbone CMVdel-mNeonGreen1xrGBD, or CMVdel-mNeonGreen-2xrGBD respectively, were digested with BsrGI and AvaI. Backbone and insert were ligated to generate CMVdel-mNeonGreen-2xrGBD and -3xrGBD.

To create different color fluorescent protein rGBD fusions, CMVdel-mNeonGreen-1xrGBD was digested with AgeI and BsrGI. The inserts dTomato, mScarlet-I and mTurquoise2 were digested with AgeI and BsrGI and ligated to the backbone. CMVdel-mNeonGreen-2xrGBD was digested with AgeI and BsrGI. The inserts dTomato was digested with AgeI and BsrGI and ligated to the backbone.

pLV-dT-2xrGBD was created by digesting the pLV backbone and the insert dT-2xrGBD with the restrictions enzymes EcoRV and ApaI and ligation of the two fragments.