StarGate® Entry Cloning via pENTRY-IBA

Step 1: Entry Cloning for Donor Vector generation

In the first step, the gene of interest (GOI) will be equipped at both termini with combinatorial sites and the LguI recognition sites, which are important for oriented insertion of the PCR fragment into pENTRY-IBA51. This is done by PCR using a proofreading polymerase.
Recombination of the PCR product with the Entry Vector at the combinatorial sites (red and orange) leads to generation of the Donor Vector. This step involves loss of the LguI restriction sites (dark orange with arrowheads), making the recombination reaction unidirectional and thereby highly efficient.

In the resulting Donor Vector the same combinatorial sites are now flanked by the Esp3I recognition sites (light blue), thereby enabling  a highly efficient and specific StarGate® gene transfer process into Acceptor Vectors in a similar manner (link: Step2).