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AssigneeInvitrogen Corporation
InventorsDavid J. Miles, Lyle C. Turner, Robert Marcil, Gina C. McConnell
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The present invention is a cell-free subcloning system utilizing three elements: (1) a donor vector that contains a nucleic acid sequence to be transferred to another vector flanked by a site-specific recombination sequence and one or more optional additional nucleic acid sequences, (2) an acceptor vector that contains a site-specific recombination sequence and one or more optional additional nucleic acid sequences, and (3) a site-specific recombinase that recognizes the site-specific recombination sequences in the donor and acceptor vectors so as to transfer the transfer sequence from the donor to the acceptor vector upon contact of the three elements of the system. Also disclosed are rapid subcloning methods employing the vectors and enzymes disclosed herein and kits for use in such methods.
A cell-free subcloning system comprising:
A method of rapidly subcloning a nucleic acid sequence, said method comprising contacting a site-specific recombinase and a cell-free solution comprising a donor vector comprising a transfer sequence flanked by a site-specific recombination sequence recognized by the recombinase, and an acceptor vector comprising at least one site-specific recombination sequence recognized by the recombinase, under conditions suitable to promote the transfer of the transfer sequence from the donor vector to the acceptor vector.
13 A method according to claim 8 wherein the site-specific recombination sequence is recognized by vaccinia DNA topoisomerase, Cre, Flp, HK022 integrase or lambda integrase.
A method according to claim 13 wherein the fusion protein producing sequence comprises an epitope-tag encoding sequence, an affinity purification-tag encoding sequence, or a functional protein encoding sequence.
A subcloning kit comprising
A kit comprising
Dependencies are inferred from the claim text.