Patent ID: 8759089

Claim:
A method for creating a recombinant cell, comprising the steps of: (a) assembling a linear targeting vector that comprises: (i) a hairpin loop at each end, (ii) a polynucleotide that encodes a gene-product-of-interest, (iii) a proximal polynucleotide containing a sequence that is homologous to a proximal part of a target polynucleotide, (iv) a distal polynucleotide containing a sequence that is homologous to a distal part of the target polynucleotide, (v) a nuclear localization signal (NLS) attached either to the hairpin loop at the end of the proximal polynucleotide or to the hairpin loop at the end of the distal polynucleotide, (vi) a first bait polypeptide attached to the hairpin loop that is not attached to the NLS, wherein the first bait polypeptide comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and (vii) a second bait polypeptide positioned between the NLS and the polynucleotide, wherein the second bait polypeptide and the NLS comprise the combined amino acid sequence of SEQ ID NO:8, (b) contacting a cell with said targeting vector, (c) selecting for the cell comprising the targeting vector in the target polynucleotide by applying a selective pressure, and (d) identifying the cell, which is a recombinant cell.