| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Anti-HaloTag Monoclonal Antibody (Mouse) (1:1000 dilution) | Promega | Cat# G9211 RRID: AB_2688011 |
| Anti-HaloTag Polyclonal Antibody (Rabbit) (1:1000 dilution) | Promega | Cat# G9281 RRID: AB_713650 |
| Bacterial and virus strains | ||
| pUG6 | Euroscarf,4 | Euroscarf: P30114 |
| pTSK405 | Addgene,2 | Addgene: 190817 |
| pTSK561 | Addgene,2 | Addgene: 190816 |
| pTSK573 | Addgene,1 | Addgene: 190881 |
| Chemicals, peptides, and recombinant proteins | ||
| Janelia Fluor 646 (JF646)-HaloTag ligand | Promega | Cat# GA1120 |
| Janelia Fluor 552 (JF552)-HaloTag ligand | Grimm et al.5 | |
| Janelia Fluor 549 (JF549)-HaloTag ligand | Promega | Cat# GA1110 |
| TMR-HaloTag ligand | Promega | Cat# G8251 |
| Yeast Nitrogen Base (YNB) powder w/o Amino Acids, Carbohydrates & w/ AS | MP Biomedicals | Cat# 4027-522 |
| CSM-TRP | MP Biomedicals | Cat# 4511012 |
| CSM-URA | MP Biomedicals | Cat# 4511-212 |
| DOBA | MP Biomedicals | Cat# 4026-022 |
| BactoTM Agar | Thermo Fischer Scientific | Cat# 214030 |
| BactoTM Peptone | Thermo Fischer Scientific | Cat# 211677 |
| BactoTM Yeast Extract | Thermo Fischer Scientific | Cat# 288620 |
| D-(+)-Glucose | Sigma-Aldrich | Cat# 8270-1KG |
| G418 Sulfate | GoldBio | Cat# G-418-1 |
| SeaKem GTGTM Agarose | Lonza | 50070 |
| Deposited data | ||
| Example single-molecule imaging dataset for transcription factor Ace1-HaloTag-JF646 acquired with 200 ms time interval (30 ms exposure) | Mendeley Data1 | Mendeley Data: https://doi.org/10.17632/hp9bwtnbgc.1 |
| Experimental models: Organisms/strains | ||
| Yeast Saccharomyces cerevisiae haploids isogenic to S288C: BY4741, BY4742 | Euroscarf6 | Y00000, Y10000 |
| Oligonucleotides | ||
| Primer for PDR5 deletion using pUG6 (KANMX) (Forward) | AGACCCTTTTAAGTTTTCGT ATCCGCTCGTTCGAAAGAC TTTAGACAAAAGCCAGCTG AAGCTTCGTACG∗ |
P101 |
| Primer for PDR5 deletion using pUG6 (KANMX) (Reverse) | AAATTCAAGAAAATTGAAA TGTAGAAAGCTCGCTGAAT TAAGAAAAAAAAGGCCAC TAGTGGATCTG∗ |
P102 |
| Diagnostic primers for PDR5 deletion check (Forward) | CTCTTCTACGCCGTGGTACG | P103 |
| Diagnostic primers for PDR5 deletion check (Reverse) | GAAGACGGTTCGCCATTCG | P104 |
| Diagnostic primer for PDR5 deletion check from KANMX gene (Forward) | GCTGGCCTGTTGAACAAGTC | P105 |
| Primer for -3×GFP tag using pTSK405 (URA3) (Forward) | -50 bp gene specific sequences upstream of the STOP codon-CAAGCGGCCGCCGCTGCTGC∗ | P106 |
| Primer for -3×GFP tag using pTSK405 (URA3) (Reverse) | -Reverse complement 50 bp gene specific sequences downstream of the STOP codon (including STOP codon)- GCGT CCATCTTTACAGTCCT∗ |
P107 |
| Primer for -HaloTag using pTSK561/pTSK573 (URA3/TRP1) (Forward) | -50 bp gene specific sequences upstream of the STOP codon-CA AGCGGCCGCCGCTGCTGC∗ |
P108 |
| Primer for -HaloTag using pTSK561 (URA3) (Reverse) | -Reverse complement 50 bp gene specific sequences downstream of the STOP codon (including STOP codon)- GC GTCCATCTTTACAGTCCT∗ |
P109 |
| Primer for -HaloTag using pTSK573 (TRP1) (Reverse): | -Reverse complement 50 bp gene specific sequences downstream of the STOP codon (including STOP codon)-CTA TTTCTTAGCATTTTTGACG∗ |
P110 |
| Software and algorithms | ||
| ImageJ | Schneider et al.7 | https://imagej.nih.gov/ij/ |
| MatlabTrack v6.0 (MATLAB) | Ball et al.2 | https://sourceforge.net/projects/single-molecule-tracking |
| TrackIT (MATLAB) | Kuhn et al.8 | https://gitlab.com/GebhardtLab/TrackIt |
| Diatrack | Vallotton et al.9 | http://www.diatrack.org/index.html |
| Sojourner | Ranjan et al.10 | https://github.com/sheng-liu/sojourner |
| Spot-On | Hansen et al.11 | https://spoton.berkeley.edu/ |
| Other | ||
| LabTek II chambers, 2 well, 1.5 mm cover glass | Nunc | 155379 |
| Falcon 14 mL round bottom PP Test tube with snap cap | Falcon | Cat#352059 |
| Microwave oven with at least 800 W power | ||
| Orbital shaker with temperature control (230 RPM, 30°C) | Thermo Fisher Scientific | MaxQ 4000 |
| Single-Molecule Imaging Microscope (HILO illumination; 100× 1.49 NA TIRF objective lens; necessary hardware and software for simultaneous two colors fast imaging (with exposure time as low as 10 ms). | Olympus | IX83 |
| EMCCD camera: iXon Ultra 888 | Andor Technology | DU-888U-CS0-#BV |
| 488 nm (100 mW) laser | Coherent | OBIS 488LX |
| 647 nm (120 mW) laser | Coherent | OBIS 647LX |
| Tabletop centrifuge with 1.5 mL, 15 mL, 50 mL tube rotors | Eppendorf | 5804R |
| Vortex mixer | ||
∗Vector specific DNA sequences are shown in blue font.