In the pFRT/LacZeo plasmid, there is an SV40 small T intron downstream of the last cDNA but before the SV40 poly A signal. What is the function of this intron? Should I remove this intron when integrating the ATG-FRT-LacZeo cassette into a lentiviral plasmid, or should I keep this intron.
It was my understanding that having an intron (and therefore an exon-exon junction after splicing) after the last stop codon, leads to nonsense-mediated decay. Any idea why this is present in this vector, as well as other (older) expression vectors?
See sequence and map here: https://www.snapgene.com/resources/plasmid-files/?set=mammalian_expression_vectors&plasmid=pFRT_lacZeo
or use the attached map file to evaluate the sequence.
Kind regards,
Jelle Bonthuis
Hi Jelle
Interesting question - I've queried this myself in the past. Haven't managed to find a decent explanation. My guess is that it's a remnant piece carried over from cloning of the original SV40 genome into plasmid vectors, as the sequence between that and the SV40pA matches that of SV40gp6 in the wild-type SV40 genome.
In terms of the other part of your question - you should omit that (and the SV40pA) from the lentiviral vector as it will be unnecessary. Lentivirus usually contains WPRE and HIV3'LTR for polyadenylation. You could keep the SV40 promoter, but even then I would select another if possible.
Best wishes
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