Example of a 4 cassettes expression vector for eukaryotic cells

What do we mean by ‘total freedom of design’? Here is a glimpse of it.

We decided to build up a vector that could drive the expression of four different proteins (4 distinct fluorescent proteins), each under the control of its own promotor, and each addressed to a different cell compartment (NucleusGolgiMitochondria and Plasma Membrane) by fusing each ORF with the appropriate addressing peptide or protein domain.

We have chosen the orientation for each expression cassette and even the location of the bacterial content, to act as buffer between the cassettes (A)

Vectors were assembled from a mix of 6 physical DNA bricks, some short containing only one genetic feature, some longer containing multiple genetic features picked from previously assembled vectors (B).

Following one single transient transfection we could observe by confocal microscopy that each protein localized in the appropriate cell compartment (C).

independant vector cassettes
DNA bricks
expression 4 distinct transgene
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