Extended Data Fig. 1: Gene-targeting to generate mutant Cdc7 alleles.

a, Verification that analog-sensitive (as) Cdc7, but not wild-type Cdc7 can be potently inhibited by 1NM-PP1. 293T cells were transfected with constructs encoding Flag-tagged wild-type (wt) or as Cdc7. Cells were treated with DMSO (Vehicle) or 1NM-PP1, Cdc7 was immunoprecipitated using an anti-Flag antibody and subjected to kinase reactions with [γ³²P]-ATP and Mcm2 as a substrate. Immunoprecipitates were also immunoblotted with an anti-Flag antibody. b, Gene-targeting strategy to generate the Cdc7^AS allele. Cdc7 exons are numbered, coding exons are in grey. M128G, as substitution; Puro, puromycin resistance cassette; 4x polyA, polyadenylation signal; red triangles, LoxP sites. c, Growth curves of Cdk1^AS/AS ESC cultured in the presence of vehicle (Control) or 3MB-PP1. d, Cdk1^AS/AS ESC treated as in c for 24h were stained with propidium iodide and analyzed by flow cytometry. Note that Cdk1-inhibited cells arrested in G2 phase, as expected. e, The principle of auxin-inducible degradation system. A plant hormone auxin recruits proteins containing the auxin-inducible degron (AID) domain to E3 ubiquitin ligase Cul1–Rbx1–Skp1–Tir1 (SCF^Tir1) for polyubiquitination and subsequent degradation by the proteasome. f, Gene-targeting strategy to knock-in the AID domain and 3xFlag-tag into the Cdc7 gene in ESC. g, Immunoblot analysis of wild-type (wt) and Cdc7^AID/AID/Tir1 (AID) ESC for expression of Flag-tagged Cdc7. h, Gene-targeting strategy to knock-in the Myc-tagged Tir1 gene into Rosa 26 locus. i, Immunoblot analysis of wild-type (wt) and Tir1 knock-in ESC for expression of Myc-tagged Tir1. Gapdh was used as loading control. j, To verify the absence of truncated, untagged Cdc7 species in Cdc7^AID/AID/Tir1 ESC, we prepared whole cell lysates (WCL) from these cells (lane 2), and probed immunoblots with an anti-Cdc7 antibody. In addition, we immunodepleted tagged Cdc7 from WCL using an anti-Flag antibody (IP-Flag) and probed immunoprecipitates (IP, lane 3) and supernatants (Sup, lane 4) with an anti-Cdc7 antibody. WCL from wild-type ESC were also immunoblotted (lane 1). We did not detect the presence of truncated Cdc7. k, To further exclude the presence of truncated Cdc7, we resolved WCL from Cdc7^AID/AID /Tir1 ESC on an SDS-PAGE gel, cut the gel into smaller fragments corresponding to different protein sizes and evaluated the presence of Cdc7 peptides in these fractions by mass spectrometry. We detected the presence of Cdc7 only in the fraction corresponding to the full-length protein. l, Gene-targeting strategy to generate Cdc7^Flox allele in ESC. Red triangles, LoxP sites. c, n=3 independent replicates; a, d, g, i, j show representative results (out of 2); c, p-values determined by two-sided t-test; error bars, SD.