Engineered HaloTag variants for fluorescence lifetime multiplexing.

General considerations Fluorophores-CA were either prepared according to literature procedures by B. Matthes or D. Schmidt (MPI-MR), kindly provided by L. Lavis (Janelia Research Campus), or purchased from commercial vendors (Supplementary Table 4 ). The synthesis of Cyanine3-CA, Cyanine5-CA, and Alexa647-CA is described in the Supplementary Methods . MaP618-Actin, MaP555-Actin, MaP555-DNA, MaP555-BG and MaP555-Tubulin were obtained from Spirochrome (SPY620 and SPY555). Fluorophores were prepared as stock solutions in dry DMSO and diluted in the respective buffer such that the final concentration of DMSO did not exceed 1% (v/v). Activity buffer (50 m m HEPES, 150 m m NaCl, pH 7.2) was used in all experiments unless otherwise stated, and 96-well plates (black, flat bottom, nonbinding (Corning)) were used unless otherwise stated. Fluorescence intensity was measured on a plate reader (Spark 20M, Tecan) equipped with filters and a monochromator. We performed excitation and emission collection as indicated in Supplementary Table 4 . Enhanced green fluorescent protein (EGFP) was excited at 485/20 nm and emission was collected at 535/25 nm.

Plasmids

A pET51b(+) vector (Novagen) was used for protein production in E. coli . Proteins were N-terminally tagged with His x10 , followed by a tobacco etch virus (TEV) protease cleavage site (ENLYFQ | G). For in vitro screening, EGFP was fused to the C-terminus of HaloTag7. A pcDNA5/FRT/TO or a pcDNA5/FRT vector (ThermoFisher Scientific) was used for transient expression as well as stable cell line establishment in mammalian cells. HaloTag7 and its variants were fused to T2A-EGFP for expression in the cytosol. HaloTag7 and its variants were fused to CEP41, H2B, TOMM20, NES, COX8, CalR/KDEL, β4Gal-T1, LAMP1, SKL, Lyn11, Ig-κ-/PDGFR, or Lifeact for expression in mammalian cells. In addition, T2A-EGFP was fused C-terminally of HaloTag7 and its variants. For cotranslational (T2A) expression of two or three HaloTags, plasmids were cloned using three different codon optimizations for HaloTag7, HaloTag9, and HaloTag11; the latter two were obtained from gene synthesis (Eurofins). The Fucci sensors were constructed as follows: Fucci(SA): HaloTag7-Geminin(1–110)-P2A-HaloTag9-Cdt(30–120), Fucci(SCA): HaloTag7-Geminin(1–110)-P2A-HaloTag9-Cdt(1–100)Cy+, Fucci(CA): HaloTag7-Geminin(1–110)-P2A-HaloTag9-Cdt(1–100)Cy– (ref. 20 ), again two different codon optimizations were used for HaloTag7 and HaloTag9. We performed cloning by Gibson assembly 24 . DNA was subsequently electroporated in E. cloni 10G (Lucigene) and plated on agar Lysogenic Broth (LB) plates with 100 µg ml –1 ampicillin and incubated at 37 °C overnight. EGFP, T2A-EGFP (Addgene plasmid no. 135443) 25 , CEP41 (Addgene plasmid no. 135446) 25 , TOMM20 (Addgene plasmid no. 135443) 25 , H2B (Addgene plasmid no. 135444) 25 , COX8 (Addgene plasmid no. 113916) 26 , Ig-κ-/PDGFR 27 and SNAPf (Addgene plasmid no. 167271) 17 were available inhouse and used as template plasmids. mCherry-LaminB1-10 was a gift from M. Davidson (Addgene plasmid no. 55069), pAAV_hsyn_NES-his-CAMPARI2-F391W-WPRE-SV40 was a gift from E. Schreiter (Addgene plasmid no. 101061) 28 , pmTurquoise2-Golgi was a gift from D. Gadella (Addgene plasmid no. 36205) 29 , LAMP1-mGFP was a gift from E. Dell’Angelica (Addgene plasmid no. 34831) 30 , hGeminin(1–110) and hCdt(30–130): pLL3.7m-Clover-Geminin(1–110)-IRES-mKO2-Cdt(30–120) was a gift from M. Lin (Addgene plasmid no. 83841) 31 and hCdt(1–100): pCSII-EF-miRFP709-hCdt(1–100) was a gift from V. Verkhusha (Addgene plasmid no. 80007) 32 ; these were all used as template plasmids. pCAGGS-Raichu-RhoA-CR, a gift from M. Lin (Addgene plasmid no. 40258) 23 , was used directly for transient transfection. For more information see Supplementary Table 18 . Plasmids generated in this work have been deposited with Addgene with accession codes listed in Supplementary Table 18 .

Protein production and purification

Proteins were expressed in the E. coli strain BL21(DE3)-pLysS. LB cultures were grown at 37 °C to an optical density at 600 nm (OD 600nm ) of 0.8, induced by the addition of 0.5 m m isopropyl-β- d -thiogalactopyranoside (IPTG) and grown at 17 °C overnight in the presence of 1 mM MgCl 2 . The cells were harvested by centrifugation (4,500 g , 10 min, 4 °C) and lysed by sonication (5 min, cycle 5, 70%, SonoPlus Bandelin). The cell lysate was cleared by centrifugation (70,000 g , 20 min, 4 °C). Proteins were purified using affinity-tag Ni-NTA (Qiagen) leading to purity higher than 95% (verified by SDS-PAGE Coomassie staining). Proteins were finally concentrated using an Ultra-0.5 ml centrifugal filter device (Amicon) with a molecular weight cut-off according to the protein size, followed by buffer exchange into activity buffer (