Does anybody have a TEV-protease-site-coding nucleotide sequence with a
commonly-used restriction site in it, preferably right at the end?
Alternatively, does some somebody know of a program to determine all
equivalent codon permutations for a small coding region, filtered for
resulting restriction site possibilities? It seems like it would be an
easy enough script to write...
Our main vectors (His- and His-MBP cleavable fusions) use blunt site right
after TEV site. This way, you are not dependent on whether the same site is
present in your gene and the cloning can be done without introducing any
artefacts (besides Gly leftover). Here is the sequence:
AGTGAAAACCTGTATTTTCAAGGCCT
AGGCCT is a StuI blunt cutter site (cheap and good enzyme) and
GGC is a Gly
So your insert is typically a PCR product that supplies the last letter in
the Gly codon and you have a complete freedom of what follows. Blunt/sticky
directional ligation, if done right, is extremely efficient and you only
need to cut PCR product with one enzyme (no need to phosphorylate primers,
the single non-ligatable joint gets repaired in E.coli).
But then, we have by now almost completely switched to QuickChange cloning
- "clone anything anywhere anytime completely artefact-free" (at least as
long as the final plasmid is not larger than ~ 9 kbp).
Dima
