Hello everyone,
so I got this plasmid which I'm supposed to use, but I need to replace GFP with another fluorescent protein, so I decided to sequence it. Because I wasn't able to make much from the first round, I have sequenced it again with more primers to cover larger part of the plasmid (also because in other plasmid I discovered many large deletions).
Anyway, one of the sequencing is very peculiar. In the bacbone_alignment.fsa file, the sequences of interest are:
pBA002a - theoretical sequence of my plasmid (without Gateway casette and GFP, which my plasmid contains)
RB_up-400 - sequencing with RB_upstream primer, reverse complement, ab1 file is attached
2a_rev - sequencing with 2a_rev primer, reverse complement, ab1 file is attached
pBA002a-R - the 2a_rev primer
old_2a_rev - sequencing with 2a_rev primer from the first round, ab1 file named pBA002_GW_GFP_rev-PREMIX is attached
So the region of peculiarity is around position 8360. There is the annealing site of the 2a_rev primer. But the 2a_rev sequence goes beyond that site (to the right, as it is reverse complement). How is that possible?
Unfortunately the upstream sequencing reaction (RB_up) was of poor quality, so only about 400 bp are usable, but they appear to overlap. Except of the first 10 nt of the 2a_rev reaction. When comparing with the old reaction, there seem to be at least 15 nt of dissimilar sequence and no annealing site for 2a_rev primer! Could there be some duplication in the plasmid? But the 2a_rev site includes the annealing site for the 2a_rev primer except of the very last nucleotide. Is that enough to prevent annealing and amplification in the sequencing reaction?
I have analogous plasmid with GFP at the N-term. I haven't noticed it before, because it didn't align so weirdly (and because the RB_up reaction was long enough to go beyond the plasmid backbone), but now I checked and it is actually analogous.
So I guess I just answered my own question *shrugging*
Now I checked the other vector (GFP-GW) and there are two NOS terminators. I guess that shouldn't be such problem for functionality of the plasmid. Or should I rather remove it?
I'm not sure how to explain these observations but, you might be better off just sequencing the entire plasmid. Plasmidsaurus can sequence entire plasmids for $15. Could resolve your entire issue and provide the highest level of sequence clarity for your downstream cloning.
https://www.plasmidsaurus.com/
Thanks for the suggestion. I was thinking about full-plasmid sequencing, but the company, where we sequence would charge I think like 800 USD for the smaller of my plasmids, so I gave up and sequenced only the most crucial part. The mystery was resolved, it is indeed double terminator, which wasn't in the original plasmid.
I don't think I would be able to justify now another sequencing. If at least it were on the same continent xD
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