Patent ID: 11905521
Assignee: THE CHINESE UNIVERSITY OF HONG KONG
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 12:
13. An isolated mammalian host cell comprising a donor construct for gene integration into a target locus within the mammalian host cell genome, the donor construct comprising (a) a coding sequence for at least one gene; (b) a polyA segment at the 3′ end of the at least one gene coding sequence; (c) an internal ribosome entry site (ires) at the 5′ end of the at least one gene coding sequence; (d) one copy of a first sgRNA target sequence located at the 5′ end of the ires, wherein the first sgRNA target sequence is about 20 to about 40 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO: 5 or its reverse complementary sequence; (e) tandem repeats of human insulator sequences located at the 5′ end of the sgRNA target sequence site located at the 5′ end of the ires; and (f) a nucleotide sequence containing multiple stop codons inserted at the 5′ end of the ires element;
wherein the donor construct is a promoterless construct;
wherein the donor construct does not comprise substantial sequence identity to the target locus within the mammalian host cell genome;
and wherein the donor construct is integrated at a 3′UTR of the target locus within the mammalian host cell genome.