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For the ORF plasmids, it states the following in the description: "The last nucleotide of the stop codon has been modified, allowing for subsequent generation of C-terminal fused proteins." How has the stop codon been modified? How can the modified stop codon be used?
The last nucleotide change of stop codon actually removes the stop codon (for example from TGA to TGC). Thus, the translation will not stop there but stop at the next available stop codon. All the ORF cDNA in our library do not have the stop codon. The removal of the stop codon will facilitate the expression of C terminal fusion protein. To use this ORF plasmid, the customer can use PCR to introduce the RE cutting sites. The customer can introduce the stop TGA at this step at the same time. If the customer wants to use the recombinant cloning way, then the customer needs to make sure the vector has its own stop codon, either right after the inserted gene or after the C terminal tag sequence.
Why is the provided vector sequence so much longer than the vector map size?
The sequence contains a 2443bp region between the AfIII and EcoRV cut site, which would be removed during the cloning process of the insert. AflII occurs at 554bp-560bp and EcoRV occurs at 2998bp-3003bp.
Why is the vector sequence online at 5kb but the vector map states a 2.6kb size?
The vector sequence contains the death gene that gets removed upon recombination with the ORF insert. The vector map is the more accurate depiction of what the vector will be with the ORF inserted.
Are any other cut sites available?
HpaI and XbaI are also useable to cut out the insert CDS from the vector, besides the stated AflII and EcoRV.