Generate fusion proteins or construct artificial operons for multiple gene expression in bacterial or mammalian cells
For several applications, it is interesting to bring two or more genes, already cloned in separate Donor vectors, into operative linkage by an intergenic region. Such an intergenic region may e.g. code for an amino acid linker sequence directly connecting, after performing the fusion procedure, the gene product of a first GOI to the product of a second GOI. Alternatively, it may include a Shine Dalgarno or an IRES site for the construction of synthetic operons in bacterial or mammalian cells, respectively.
Fusion Cloning Procedure
StarGate® fusion cloning is an optional intermediate step between the Entry reaction and the Transfer reaction. It allows easy and fast fusion of two genes of interest (GOI-1 and GOI-2) present in separate Donor Vectors by performing two sequential StarGate® subcloning reactions.
The first subcloning reaction is placing each GOI into a special Fusion Vector. One Fusion Vector (pNFUSE-IBA-derivative specifying the intergenic region) is designed for upstream, while the other (pCFUSE-IBA) is for downstream positioning of the respectively inserted GOI. The Downstream Fusion Vector is identical in each reaction. Upstream and downstream Fusion Vectors with inserted GOI are then assembled in a directed manner within pENTRY-IBA20 thereby providing a new Donor Vector consisting of GOI1 fused to GOI2 by the intergenic region of the given pcFUSE-IBA derivative.
The fused GOIs are now ready to be subcloned into any of the StarGate® Acceptor Vectors available for expression ("transfer reaction"). Upstream Fusion Vectors with different intergenic regions are included as part of our new Fusion Cloning Sets. |
