For rapid cloning of genes of interest into a yeast expression vector, the GatewayTM technology can be used.
Method Article
Generation of pDRf1-GW
https://doi.org/10.1038/nprot.2007.130
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For rapid cloning of genes of interest into a yeast expression vector, the GatewayTM technology can be used.
pDRf1-GW is a yeast shuttle vector carrying a GATEWAYTM cloning cassette (Invitrogen, Carlsbad, CA) and an f1 viral replication origin. Generate pDRf1-GW by substituting a SpeI-SalI fragment from pDR1961 with a new multi-cloning sequence (SpeI, EcoRI, NotI, PstI, SalI).
Amplify by PCR a 500bp DNA fragment containing the f1 replication origin from pGEM-T-easy (Promega, Madison, WI). Use primers containing a HindIII restriction site, and then clone into the HindIII site located upstream of the PMA1 promoter fragment to generate the pDRf1 vector.
Amplify the GATEWAYTM fragment by PCR with primers containing an EcoRV site and clone in a blunt-ended NotI site in pDRf1 to generate pDRf1-GW.
Rentsch, D. et al. NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis. FEBS Lett. 370, 264-268 (1995).
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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