Gap between promoter and gene required ??

Model Organism: Worms Scientific Discussion
1

Hello !!!
I am cloning my gene in front of RAB3 (neuronal promoter).
Should I clone my gene directly after end of promoter sequence OR leave a spacer or a gap in promoter and gene ?
In some plasmids I have, there is 50-100bp gap.
Any experience ???

thank you,
Regards,
Anand (Postdoc) - UCSF

2

I usually clone a promoter up the the ATG start codon, and clone my gene immediately after the ATG in frame (mainly Gibson cloning). Some of the extra sequence could be remnants of the cloning approach (restrictrion enzyme cutting and ligation, or Gateway) or some sort of enhancer-based expression system, where the regulatory element of interest in used with a minimal promoter like (delta)pes-10. For tissue-specific drivers, I use the convention discussed at the beginning of the message. But maybe other folks have specific cases where a gap is useful or required.

3

I guess it depends on what the gap is, where it came from, and why it’s there. Many of the Fire lab vectors have an artificial intron that enhances expression, so I would definitely keep that there if that’s the only intron in your sequence. If you don’t know what it is, you should figure that out because it might have an ATG in there (in any of the 3 frames) and that could lead to aberrant expression of not-your-protein/gene.

-Kevin.

4

Hi Anand,

I have cloned rab-3 both without gap and AvrII in between promoter and gene. In both cases, expression is fine and Pan-neuronal.
So, until and unless you have a reason to add linker, keep it gap less.
Good Luck!
Anuj

5

What Jordan said. Additionally, while worms do not have a Kozak sequence, they do bias towards a string of As before the ATG. You’ll find an “AAAAG” in front of the first ATG in many fire vectors. If I have the choice, I still this in there (in frame if relevant) just to make sure I get good expression.Maybe that’s just superstition. :stuck_out_tongue: