US 5,965,444 AGranted

Method for constructing transformant not having selective marker gene

AssigneeSuntory Limited

InventorsToshihiko Ashikari, Hiroto Kondo, Keiko Sakakibara, Hiroyuki Araki, Yasuji Oshima

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Abstract

A DNA construct wherein a DNA fragment which is recombinable in yeast chromosomal DNA is directly or indirectly linked at both ends of a DNA fragment which comprises a pair of R sensitive sequences oriented in the same direction and flaking both an R gene placed under the control of an inducible promoter and an expressible selective marker gene, which is a DNA construct designed with the R sensitive sequences non-symmetrically shortened, so that no functionable R sensitive sequence remains after the R sensitive sequence recombination has occurred by expression of the R gene and the selective marker has been removed. Since no functionable R sensitive sequence remains after removal of the selective marker, recombination does not occur again, and thus the same selective marker may be used for multiple insertions of foreign genes.

Claims10

Dependent claims

A DNA construct comprising an R gene positioned under the control of an inducible promoter, and an expressible selective marker wherein the R gene and the selective marker are flanked by a pair of R sensitive sequences oriented in the same direction so as to form a removing unit which is directly or indirectly flanked by a pair of DNA fragments capable of recombination with a chromosomal DNA, wherein each of said R sensitive sequences comprises the following nucleotide sequence:

5'-TTGATGAAAGAA TACGTTA TTCTTTCATCAA-3' inverted spacer inverted repeat (1) sequence repeat (2)
or a sequence substantially identical with said nucleotide sequence,
wherein the R sensitive sequence located nearest said R gene lacks between 1 and 10 nucleotides at the end distal from the spacer sequence in the inverted repeat which is at the opposite end from the end adjacent to said R gene, and the R sensitive sequence located nearest said selective marker gene lacks between 1 and 10 nucleotides at the end distal from the spacer sequence in the inverted repeat which in at the opposite end from the end adjacent to said selective marker gene.

Dependencies are inferred from the claim text.