(A) Schematic representation of the applied strategies for improved cell cycle tag methodology. Upper panel: conventional cell cycle tag methodology was limited by only one construct for each cell cycle phase. Lower panel: the advanced cell cycle tag toolbox was expanded to 46 constructs. Therefore, we used new promoters and degrons from Clb5 and Clb1, chimeric promoter-degron combinations and protein expression was crippled using 5´UTR truncations and upstream out of frame ATGs. Vertical bars indicate location of cell cycle regulatory elements in the promoter and N-terminal degron sequence (see Figure 1—figure supplement 1 for detailed description of the tagging procedure). (B) New cell cycle tag constructs allow cell cycle-restricted expression of GFP at varied peak expression levels. Anti-FLAG westerns of cells expressing Clb5pClb5-, Clb6pClb5-, Clb6pClb6-, Clb1pClb1-, Clb2pClb1-, Clb2pClb2-3FLAG-tagged versions of GFP after G1 arrest with α-factor and synchronous release through the cell cycle up to the next G1 phase. Pgk1 western was used as control and DNA content measurements indicate cell cycle progression at the individual time points below (see Figure 1—figure supplement 2 for G1 release experiments of the corresponding 5´UTR truncations and for constructs containing upstream out of frame ATGs). (C) New promoters and degrons, chimeric promoter-degron combinations, 5’UTR truncations and upstream out of frame ATGs allow a broad spectrum of peak expression levels of cell cycle-restricted GFP. Western blot analysis of peak expression levels of cell cycle-tagged 3FLAG-GFP variants at indicated time points after G1 release (20 min = S phase, 50 min = M phase, 100 min = G1 phase). DNA content measurements below indicate cell cycle progression. Graph: peak expression levels were quantified using Image-J and signals of the individual constructs were divided by the corresponding Pgk1 sample to normalize to overall protein levels (see Figure 1—figure supplement 3 for an overview of all cell cycle-tagged GFP versions in cells arrested in the corresponding cell cycle phase). (D) Schematic representation of the suggested cell cycle tag strategy using two sets of constructs with matching ‘low’ and ‘high’ peak expression levels. ‘Low’ expressing constructs (light colours) are chosen by matching peak expression levels similar to the endogenous protein but will show underexpression at cell cycle phase transitions. ‘High’ expressing tags generally show higher expression compared to the wildtype protein with the advantage of broader timeframes of action (timeframes in which protein levels are similar or higher than endogenous protein levels).