Hi every body,
Plasmid pIRES-hrGFP1a is a product of Stratagen. My link
In this vector there is a MCS for inserting the gene before a FLAg tag.
In the vector manual you will see this sentence: The presence of stop codons in-frame with the 3× FLAG tag must be considered when inserting genes into the MCS. it says you have to put your insert which has the stop codon before 3× FLAG tag. If so, translation will be terminated before Flag tag, how the FLAG tag peptide can be fused by our interested protein as there is a stop codon before the FLAG tag?
I need the squence of this vector but the company provides the map, not the sequence. Does anyone have the sequence information of vector pIRES-hrFGP-1a?
You do not need the sequence of the whole plasmid. What you need to do is you have to use any of the following restriction enzymes for digestion (sites for them are downstream of the stop codon, so you will cut it out): SmaI/XmaI, BamHI, EcoRI, SphI, SalI, XhoI. Of course, these would be enzymes on the 3' end of your insert, you still have to remember to remove any possible stop codons from your insert, and also to ligate in-frame with the FLAG tag. Also, the question is if you want to clone in directionally (choosing a different restriction enzyme on the 5' end) or adirectionally.
Thanks a lot , very useful, I need the sequence as I want to make a recombinant plasmid using this plasmid. the company does not provide the sequence
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