Depends entirely on whether you are working in a prokaryotic or eukaryotic system. While you can clone into an intermediate cloning vector, this can be "extra" work if your ultimate goal is expression. Please comment on what type of system you are working in.
Some additional info that may be relevant for mammalian expression systems:
1) SpeI is found in many commonly used promoters such as PGK and some versions of the CMV promoter (such as in pCDNA3.1). This may limit its utility. On the other hand, SpeI produces a compatible sticky end with NheI or XbaI, so you may be able to use these sites instead as acceptor sites at the 3' end of your vector.
2) NcoI is found at many start sites of eukaryotic cDNAs that have the Kozak sequence ACC_ATG; if the second codon is Gnn, you will encounter a NcoI site. As a consequence, many mammalian expression vectors that contain other gene cassette (a GFP reporter, or SV40-antibiotic resistance gene, for example) are likely to have one or more NcoI sites separate from the MCS, that will limit its utility as a cloning site.
Depends entirely on whether you are working in a prokaryotic or eukaryotic system. While you can clone into an intermediate cloning vector, this can be "extra" work if your ultimate goal is expression. Please comment on what type of system you are working in.
Some additional info that may be relevant for mammalian expression systems:
1) SpeI is found in many commonly used promoters such as PGK and some versions of the CMV promoter (such as in pCDNA3.1). This may limit its utility. On the other hand, SpeI produces a compatible sticky end with NheI or XbaI, so you may be able to use these sites instead as acceptor sites at the 3' end of your vector.
2) NcoI is found at many start sites of eukaryotic cDNAs that have the Kozak sequence ACC_ATG; if the second codon is Gnn, you will encounter a NcoI site. As a consequence, many mammalian expression vectors that contain other gene cassette (a GFP reporter, or SV40-antibiotic resistance gene, for example) are likely to have one or more NcoI sites separate from the MCS, that will limit its utility as a cloning site.
You should check out the www.addgene.database. You can call them if you can't find what you need. In particular this part of the site might be helpful: http://www.addgene.org/empty_backbones/
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology