Recombinant yeast and preparation method thereof
A yeast and recombinant enzyme technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial-based methods, etc., can solve problems such as easy loss, low expression level, and inability to strictly control the efficiency of exogenous promoters. Achieve the effect of low cost and simple operation
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[0043] Embodiment 1, the construction of recombinant yeast
[0044] 1. Construction of expression vector pKGAPGA-amy-18S
[0045] (1) Construction of the vector pGAPGA: construction methods such as literature (Houhui Song, Yong Li, Weihuan Fang, Yunfeng Geng, Xu Wang, Min Wang, Bingsheng Qiu. Development of a set of expression vectors in Hansenula polymorpha. Biotechnology Letters, 2003, 25 (23): 1999-2006.). (Institute of Microbiology, Chinese Academy of Sciences)
[0046] (2) Construction of vector pKGAPGA
[0047] In order to be able to use the loxP site and the Cre recombinase system to knock out the resistance screening marker gene sequence on the vector pGAPGA, a pair of primers loxP-F and loxP-R were designed. The nucleotide sequences of the primers are as follows:
[0048] loxP-F:
[0049] 5'-GataacttcgtataatgtatgctatacgaagttatAGATCTAGGAAAAGGTGACAACTCGCAAAGT-3' (sequence 5 in the sequence listing)
[0050] loxP-R:
[0051] 5'-TataactcgtataatgtatgctatacgaagttatGGA...
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