While sequencing my pGEX-6p1 vector for protein expression, I discovered an insertion in the origin of replication—five consecutive 'T' residues instead of the original four. As a result, my Ori region is 590 bp instead of 589 bp. Despite this variation, plasmid yield remained consistent when isolated from the same amount of cell pellet. Should I proceed with protein expression using this vector, or would it be advisable to sequence additional samples to confirm the exact Ori sequence? I appreciate your insight.
That extra 'T' likely won't affect the expression of your protein. If you're getting good plasmid yields, I wouldn't worry too much about such a small modification. It could even be a sequencing error, as Sanger sequencing can sometimes struggle with runs of more than four repeated nucleotides.
Thank you @Daniel Martin. The whole plasmid was sequenced by Eurofin Genomics via Nanopore sequencing technique. I will keep posted how the expression goes.
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