Hello. I need to clone a gene into a vector. Gene must be merged with His tag at protein N-terminus.
I am trying to clone with program SnapGene.
This is my first time even trying to clone something into a vector and I have a few problems.
1. How do I know where actually is my N-therminus in a gene. I know I can translate gene, but the protein sequence itself gives me 0 information where is C or N end.
2. Now for example I believe my N-therminus is near the green box (above link). I should cleave somewhere at this point and then
his tag. The problem now would be the terminal stop conod, which would not let me translate his tags.
So this approach in general is bad?
My second idea is to use my own primer with 6xHis and ignore those 6xHis which are on my vector. Then I would end with:
Any suggestion and ideas how to solve this problem?
Edit: I did some research.
I believe I can cleave at NspI (first picture - my gene) and add CAT-CAT-CAC-CAC-CAC-CAT. I would end with market N-terminus gene. But now how do I insert it into vector? + It would leave sticky ends?
Edited by Dancia, 08 November 2014 - 06:02 AM.