I have cloned a gene with EcoRI/BamHI restriction sites into p3XFLAG-CMV-14. The sequencing has confirmed that the gene has been inserted in the right direction. For some reason, I would like to reverse the direction of the gene in the same vector backbone. Is there any method to do it?
Thanks in advance!
Check if the MCS of your backbone is flanked by two restriction sites of the same type. You can then digest and re-ligate and some will have the MCS+insert flipped.
Robert Adolf Brinzer thanks for the suggestion. But there is no similar restriction sites flanking the MCS of my backbone. What else can I do?
You could PCR your insert and add a different set of restriction sites on the PCR primers so that you can clone it in reverse orientation.
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