the-thought-emporium
commented
3 years ago Unfortunately I've already sent it off for synthesis. I wish this had been caught sooner. It's still very fixable, it's just annoying. Would need to remove the NdeI site from the backbone. Luckily you can get humanized mice, so there isn't as pressing of a need to sub out the FC domain, so we'll start with that. But if it's an issue, fixing the backbone shouldn't be too expensive
UWKFS
I was wondering if the sequence posted as the V3 is the up to date sequence since it has been a while since upload? I was looking through the sequence and there are 2 NdeI cut sites, (the one you added and one on the AAV backbone) is this something that has been fixed previously?
If not fixed it may be a good switch since, if you wanted to swap the human IgGA FC to another FC. For example, to a mouse IgG for testing in mice (I am relatively sure that human FC's won't work in mice but I could be mistaken) then it will be much easier to just synthesis the mouse IgG and clone it after removing the human IgGA. As the plasmid stands now you will have to either remake your insert with a mouse IgG or cut your plasmid with BspEI and have your insert repair the rVAR2 C-terminal sequence with a new IgG FC.
If I am correct, hopefully, you haven't ordered the synthesis of this insert yet but either way, I hope this helps!
PS. I am currently doing my MSc in Plant Biochemistry and your videos on benching have helped me save so much time. So thanks for posting these! and keep up the good work