Patent ID: 9102944
Filing Date: 2015-08-11
Classification: C12N

Abstract:
1. A composition comprising (i) a first polynucleotide having a first strand and a second strand, wherein said first polynucleotide is a first vector that comprises (a) a stuffer sequence, (b) a first topoisomerase recognition sequence and a second topoisomerase recognition sequence, (c) a first nicking agent recognition sequence and a second nicking agent recognition sequence, and a (d) selectable marker, (ii) a sequence-specific topoisomerase that reversibly cleaves a single strand of double stranded nucleic acid of the first polynucleotide at said first topoisomerase recognition sequence or said second topoisomerase recognition sequence, and (iii) a second polynucleotide, wherein the first topoisomerase recognition sequence and the first nicking agent recognition sequence comprises the sequence CCCTTNNNNGACTC (SEQ ID NO: 23) or GTGTCGCCCTTATTCGATCC (SEQ ID NO: 43), such that the first topoisomerase recognition sequence is in said first strand and said first nicking agent recognition sequence directs a nicking agent to nick said second strand of said first vector, the second topoisomerase recognition sequence is in within fifty nucleotides of the second nicking agent recognition sequence, the second topoisomerase recognition sequence is in said second strand and said second nicking agent recognition sequence directs a nicking agent to nick said first strand of said first vector, the stuffer sequence is between said first topoisomerase recognition sequence and said second topoisomerase recognition sequence, the stuffer sequence comprises a counter-selectable marker, a double stranded break site, a gene that encodes a fluorescent protein, or a gene that encodes a protein that participates in catalyzing a chromogenic reaction, said first vector is nicked in the first strand, in or close to said second nicking agent recognition sequence, and said first vector is nicked in the second strand, in or close to said first nicking agent recognition sequence, by a sequence-specific single-stranded nicking endonuclease.