Mirfp670

The plasmids pBAD-(His₆)miRFP670 (Addgene no. 79984),^{29 (link)} pBAD-(His₆)miRFP709 (Addgene no. 79986),^{29 (link)} and pWA PAS-E BAD K-GAFm (Addgene no. 39866)^{25 (link)} were gifts from Vladislav Verkhusha. All plasmids were verified by DNA sequencing.

Gibson assembly, Gateway cloning, and standard molecular biology were used for DNA cloning. Human RAB34 cDNA was obtained from GE Healthcare (MHS6278-202807876) and cloned into Gateway pENTR plasmid pDONR221 (Invitrogen); RAB34 mutants were prepared by site-directed mutagenesis of pENTR-Rab34. Plasmids for tetracycline-inducible expression of LAP-Rab34 (LAP consists of GFP, TEV protease site, and S-tag) were constructed by modification of pCW-Cas9 (Addgene #50661, gift from Eric Lander and David Sabatini). Stable expression of fluorescently tagged ciliary and centriolar proteins was achieved by modification of pCW-Cas9 using cDNAs for the PGK promoter, ARL13B (Addgene #40879, gift from Tamara Caspary), EHD1 (gift from Chris Westlake), Rab8 (gift from Maxence Nachury), and Centrin2 (gift from Tim Stearns), miRFP-670 (Addgene #79987, gift from Vladislav Verkhusha), and mScarlet-I (Addgene #85044, gift from Dorus Gadella). Plasmids for CRISPR-based knockout were constructed using pMCB320 (Addgene #89359, gift from Michael Bassik) and pMJ179 (Addgene #89556, gift from Jonathan Weissman); see Key Resources Table for sgRNA sequences. Plasmids for transient transfection of RAB34 were made by Gateway cloning using pEF5-FRT-LAP-DEST^{55 (link)},^{56 (link)}; for bacterial protein expression, RAB34 variants were cloned into pGEX-6P1 (GE Healthcare).