OPRM1_pGHE FLAG Tag

OPRM1_pGHE with FLAG tag SnapGene file: https://drive.google.com/file/d/1W6GsXY_hKTeBMXpDNQK6mNYcYH5JFDjC/view?usp=sharing

Plasmid maps of OPRM1_pGHE and FLAG:

The left image is the plasmid map for human mu opioid receptor in the pGHE plasmid with the FLAG epitope tag inserted in the N-terminus of the receptor. The FLAG tag was inserted after the methionine amino acid at the N-terminus. See the link above for the full sequence which is in a SnapGene file that has been uploaded to the Norimatsu Google Drive under the Summer 2021 info folder.

The image to the left is the map of the human mu opioid receptor in pGHE without the FLAG tag. This image is included for comparison to the image above that contains the FLAG tag.

Sequence maps of OPRM1_pGHE and FLAG:

Below are images of the sequences for hOPRM1 in pGHE + FLAG and hOPRM1 in pGHE. These images were taken for comparison of the two sequences to show the difference made by the insertion of the FLAG tag. As mentioned above the FLAG tag is inserted after the methionine amino acid of the N-terminus. Restriction enzyme sites that could be used for synthesis are SmaI and AfeI.

hOPRM1_pGHE + FLAG

hOPRM1_pGHE

Reference Materials:

Trapaidze, N., Gomes, I., Cvejic, S., Bansinath, M., & Devi, L. A. (2000). Opioid receptor endocytosis and activation of MAP kinase pathway. Brain research. Molecular brain research, 76(2), 220–228. https://doi.org/10.1016/s0169-328x(00)00002-4
Gupta, A., Rozenfeld, R., Gomes, I., Raehal, K.M., Decaillot, F.M., Bohn, L.M., Devi, L.A. (2008). Post-activation-mediated Changes in Opioid Receptors Detected by N-terminal antibodies. The Journal of biological chemistry, 283(16), 10735-10744. https://doi.org/10.1074/jbc.M709454200

In the two reference materials above, the mouse mu-opioid receptor was used and tagged with FLAG. Both publications have schematic representations of the mouse mu-opioid receptor in which they show that the FLAG epitope tag was placed before the methionine amino acid (in the schematic). This placement was shown to be effective by the different experimental procedures in the two papers.

To ensure that this placement could translate to the human opioid receptor, an alignment between the mouse and human OPRM1 was done. The two are approximately eighty percent similar, and based on the rotation of the helix, the placement of the FLAG tag will not affect the structure of the helix. A screenshot of the alignment is shown below for reference:

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