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StarGate® Cloning Principle in Detail |
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Step1: Donor Vector generation |
The gene of interest (GOI) has to be equipped in a first PCR-step at both termini with combinatorial sites and the StarCombinase1™ recognition area which are important for oriented insertion of the PCR fragment into pENTRY-IBA51 Recombination of the PCR product and the Entry Vector at the combinatorial sites (red and orange) leads to generation of the Donor Vector under loss of all StarCombinase1™ binding areas (dark orange with arrowheads) making the recombination reaction unidirectional and thereby highly efficient. In the resulting Donor Vector the same combinatorial sites are now under control of StarCombinase2™ (light blue) thereby enabling a highly efficient and specific StarGate gene transfer process into Acceptor Vectors in a similar manner (Step2). NEW Combi Entry Cloning Set (for appropriate primers use StarPrimer D´Signer Software) ** **Please note IBA has changed the Entry Cloning Step by May 2010. If you have used StarGate in the past please check the sequence of your Entry Cloning Primers. |
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In the second step, the GOI will be transferred from the Donor Vector into Acceptor Vectors providing the desired genetic surrounding (i.e. tag, promoter, signal sequence etc.) by means of Star-Combinase to create corresponding Destination Vectors. The desired E. coli clones carrying a Destination Vector can easily be identified through blue/white selection (see StarGate data Fig. 2). The Destination Vector of this example places the GOI under control of the CMV promoter allowing GOI expression in mammalian cells. In addition, a tag is fused to the C-terminal end of the GOI expression product. |
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