I cloned a gene of interest in-frame with the 3XFLAG sequence (C terminus) to pcDNA 5FRT/TO vector. The gene of interest has the start codon and the FLAG tag has the stop codon so that it can be expressed as a fusion protein for pull down experiment.
I also have the vector only with the FLAG sequences cloned. However, this vector do not contain the ATG start site. I do need this vector only with the FLAG expression for use as a control in my experiment. Any ideas to insert ATG codon to this FLAG only containing vector?
...Can do the usual PCR amplification of FLAG sequence including ATG in the forward primer then followed by Restriction digestion, ligation and cloning. Are there any better ways to perform this?
Hi there,
An alternative is to clone in one upstream unique site of the vector (very close to the FLAG tag) a dsDNA fragment obtained by hybridization of two 25-30mer oligonucleotides (hybridization generating ends compatible with vector for ligation and sequence containing an ATG codon in frame with FLAG tag). If doing this in one single site, half of the ligation products only will be OK. If you don't want to be confronted to this issue, use two upstream unique sites for cloning.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning