I have sequenced the different colonies with the forward primer (T7 promoter primer) and getting the same result whole gene with N-terminal His tag..But I was not getting any expression so did sequencing again but with Rev Primer.. I got different sequencing result with gaps and different codons...
Nagarjuna Reddy Pamudurthy
Hi,
you can clone the same gene into any simple vector and check for expression if it works then do sub clone in to your vector of interest and check for expression which may works, but i am still not sure why you got the sequencing errors while you using reverse primers for sequencing the same plasmid while it is good with T7 forward primer.
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