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Quantitative PCR To analyze mRNA expression, total RNA was collected from cells using the PureLink RNA Mini Kit (Thermo Fisher Scientific), and 1 g was reverse-transcribed using the ProtoScript II First Strand cDNA Synthesis Kit with oligo(dT) primers and murine RNase inhibitor (New England Biolabs)

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Results A counter-selection system was engineered in Saccharomyces cerevisiae based on the known feedback inhibition of gamma-glutamylcysteine synthetase ( GSH1 ) gene expression, which is rate limiting for GSH synthesis: the GSH1 ORF and the counter-selectable marker GAP1 were expressed under control of the TEF1 and GSH-regulated GSH1 promoters, respectively