Figure 4

Design of vectors for random heterologous expression with the gene’s natural promoter (A), with an alternative promoter (B), for targeted gene replacement (C) and in locus overexpression (D). A) Expression of the gene of interest from a random locus in the genome, driven by the gene’s natural promoter. Note the use of the B2e USER-Brick to allow for direct fusion of the selection marker cassette with the B2 vector backbone. B) Overexpression of the gene of interest from a random genomic locus, with the expression driven by a heterologous promoter, in this case the gpdA promoter from Aspergillus nidulans. Note the use of the B2e USER-Brick to allow for direct fusion of the selection marker cassette with the B2 vector backbone. C) Replacement of the gene of interest. Note that the HRS1 fragment can also be reused for in locus overexpression experiments. D) In locus overexpression of the gene of interest by targeted integration of a strong constitutive promoter. Note that the HRS1 fragment can be reused for deletion experiments. Primers are represented by solid black arrows. Aberrations: gDNA = genomic DNA; P = promoter; CDS = coding sequence; T = terminator; RB & LB = right & left borders defining the T-DNA region; T-DNA = transfer DNA.