Fig. 2.
Combinatorial assembly of ATF/BS or CDS units in Entry vector X. a Combinatorial insertion of JUB1-derived ATF/BS units in Entry vector X. Three-partite fragments, harboring an IPTG-inducible promoter, the ATF, and the CYC1 terminator are PCR-amplified from expression plasmids NLS-DBDJUB1-GAL4AD (light blue arrows), NLS-JUB1-EDLLAD-EDLLAD (blue arrows), and NLS-JUB1-GAL4AD (dark-blue arrows)4. Two synthetic promoters, harboring two (two light-blue squares) and four (four light-blue squares) copies of the JUB1BS, are PCR-amplified from reporter plasmids4. ATFs and BSs amplicons are mixed in 1:10 molar ratio and cloned into FseI/AscI-linearized Entry vector X. The diagram shows six possible outcomes. The three required JUB1-derived ATF/BSs, NLS-DBDJUB1-GAL4AD/2×, NLS-JUB1-EDLLAD-EDLLAD/4×, and NLS-JUB1-GAL4AD/2×, are numbered 1–3, correspondingly. Brown bent arrow, IPTG-inducible promoter. Brown “T”, yeast CYC1 terminator. b Gel electrophoresis to identify required JUB1-derived ATF/BSs. The verification of construct numbers 1–3 was done using colony PCR on ATF/BS fragments. M1: HyperLadder 1 kb (Bioline). c Insertion of CDS units into Entry vector X. CDSs (light-orange arrows) with rare RE sites (blue squares; iii: AsiSI, a: I-CeuI, b: I-SceI, c: PI-PspI, d: PI-SceI/AscI) in their 5′ regions, a yeast terminator (brown “T”) carrying similar RE sites (blue squares) in the 3′ region, and promoters of E. coli marker genes (green bent arrow) fused to Z1, Z2, Z3, Z4, or Z5 are cloned into PacI-digested Entry vector X. The Z and RE sites define based on Level 1 vectors (Destination vectors I/II: Z1 and iii; Acceptor vectors A/E: Z2 and a; B/F: Z3 and b; C/G: Z4 and c; and E/F: Z5 and d). The HRs X0, Z0–Z5, and Y0 are explained in footnote to Supplementary Data 3