FIGURE 4: Correct intracellular localization depends on the promoter strength used for expression of the fusion proteins. (A) Schematic representation of the plasmid showing the different MoClo cassettes. The different promoters used are indicated in green. (B) Wild type (YPH499) cells with the different expression plasmids were grown to mid log phase in synthetic glucose medium. For the asterisk-labeled GAL1 sample, cells were grown on lactate-containing medium to mid-log phase; then 0.5% galactose was added, and cells were further grown for 4h. Cells were harvested, and the fluorescence intensity of the NeonGreen protein was measured by fluorescence spectroscopy in a plate reader. For each biological replicate (N = 3) technical triplicates were measured. Shown are the mean values from all three independent measurements, error bars represent the standard deviation. (C) Representative fluorescence images showing the distribution of NeonGreen in the different strains indicated. Samples in which the protein distribution showed the correct intracellular compartments were labeled by red frames and check marks. Scale bar, 5 µm.