I have to use the gateway system to insert the expression cassette (the promoter, flag-tagged ORF, and the 3’UTR) into the MOSSCI vector pCFJ150, (Here is the map of pCFJ150:http://tinyurl.com/m9hc8v). But I can’t find any suitable entry clones for insertion in the website of invitrogen (http://www.invitrogen.com/site/us/en/home.html).
Could anyone give me some suggestions and advices about adapting the expression cassette into pCFJ150 with multisite gateway system?
If I understand your question correctly, then you are looking for the entry vectors that you need to clone the promoter, the ORF and the 3’UTR into?
The pCFJ150 vector is compatible with the three-fragment gateway kit from invitrogen (search for catalogue number 12537-023 at invitrogen’s website, or google it). The promoter is typically cloned into pDONRP4-P1R), the ORF into pDONR221 or pDONR201 and the 3’utr into pDONRP2R-P3.
The manual is pretty good at explaining the basics of multi-site gateway. And with a bit of luck someone at your institution already has the three entry vectors so you don’t need to buy them.
If you already have an expression construct (promoter::FLAG-ORF::3’utr) it may be easier just to subclone it into pCFJ151 which has an MCS, instead of gateway sites.