Overexpression library construction and screening. (a) Construction of an HA-tagged vector. The pCMha190 vector used here was constructed by insertion of a linker (gray box) in place of the multiple cloning site in vector pCM190 . Features shown include the promoter and TATA box as well as the terminator from the original plasmid (open boxes), and the start codon, HA-tag, BamHI site and stop codons (thick vertical bars) from the introduced linker sequence. The linker was composed from the following annealed oligonucleotides: EXP3: 5'-GATCGTTTAAACCATATGTACCCATACGACGTCCCAGACTACGCTGG ATCCTGACTGACTGATC-3', EXP4: 5'-GGCCGATCAGTCAGTCAGGATCCAGCGT AGTCTGGGACGTCGTATGGGTACATATGGTTTAAAC-3'. (b) Library construction in pCMha190 (see Materials and methods for experimental details). The resulting ligation product is schematized, with the insert as a striped box and adaptors as hatched boxes. Sequences shown below are from junctions, with uppercase letters corresponding to vector (the extra nucleotide from filling-in is underlined), lowercase letters to adaptors and bold nnn's to insert. Arrows indicate the different primers used: SEQ8 and SEQ4 are used for PCR amplification of the insert, and SEQ1 for sequencing (see sequences in Additional data file 8). (c) First-round screening of toxic phenotypes. The growth of random and control clones on selective medium in uninduced and overexpression conditions is shown. Drops of serial dilutions (1/100 to 1/100,000) of cultures were grown for 45 h at 30°C. A3, non-toxic control clone transformed by pCMha190; H1, toxic control clone transformed by MCM1 gene cloned in pCMha190; G1, B2, D2, E3, library transformed clones, exhibiting different levels of toxicity in overexpression conditions (see Figure 2).