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Figure 1 | Microbial Cell Factories

Figure 1

From: Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae

Figure 1

Generic plasmid vector map for the pCEV dual gene cassette expression vectors. Each plasmid has two expression cassettes, each driven by a different promoter (P1 or P2), and a selection cassette with either aphA1 or ble genes. Promoter/selection combinations are shown in Figure 1. Each gene of interest can be optionally tagged using either a FLAG epitope tag (Expression Cassette 1) or a c-myc epitope tag (Expression Cassette 2). The selection cassette is controlled by the TEF promoter and terminator from Ashbya gossypii (P AgTEF1 and T AgTEF1 ). The terminator for Expression Cassette 1 is derived from the yeast alcohol dehydrogenase (AHD1) gene. The terminator for Expression Cassette 2 is derived from the yeast cytochrome C (CYC1) gene. The potential exists for integration of the expression cassettes onto the chromosome by amplification using primers with homologous arms; in this case, the selection marker cassette can be removed to recycle the marker via CRE-mediated recombination at the available loxP sites. A pUC origin of replication (Ori) is available for maintenance in E. coli and a 2 μm Ori for maintenance in S. cerevisiae. Selection in E. coli can be performed using either the aphA1/ble selection cassette, which is functional for selection in E. coli, or using the beta-lactamase (bla) ampicillin resistance gene. The dual promoter region is not to scale (size varies depending on which promoters are present) and the selection marker is also not to scale (can be either aphA1 or ble). Different vector components in these two regions, as well as the unique restriction enzyme sites found in each multiple cloning site (MCS), are shown in Table 2. Note that the MCS spans the epitope tag sites for each expression cassette; choice of restriction sites will determine whether or not the tag sequence is retained in the construct.

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