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An in vitro recombination method to convert restriction‐ and ligation‐independent expression vectors
Author(s) -
Guo Feng,
Chiang MingYi,
Wang Yingtong,
Zhang YuZhu
Publication year - 2008
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200700170
Subject(s) - expression cassette , recombinase , in vitro recombination , vector (molecular biology) , restriction enzyme , recombination , computational biology , biology , cloning (programming) , restriction site , multiple cloning site , flp frt recombination , genetics , site specific recombination , cloning vector , expression vector , computer science , molecular cloning , recombinant dna , dna , gene , genetic recombination , gene expression , programming language
In recent years, restriction‐less recombination cloning systems based on site‐specific recombinase with high efficiency have been proven to be very successful. Thus, it is desirable to convert existing conventional vectors to recombination vectors. In this report, we describe the conversion of a set of widely used conventional vectors to Gateway® recombination expression vectors. An att B cassette flanked by several restriction enzyme sites was inserted in a cloning vector, and then subcloned into existing vectors to be converted to construct intermediate vectors containing the att B cassette, which were then converted to recombination expression vectors by in vitro recombination. The intermediate vectors generated in this study can be used for releasing the att B cassette to convert other vectors using the same protocol described here. With the increasing number of recombination vectors constructed with this protocol, the likeliness of releasing the att B cassette from an existing vector, rather than synthesizing it with PCR, will increase. The final expression vectors can also be used for releasing the att R cassette for constructing new vectors.