Table 3.

Primer sequences used for the cloning of mouse μ-opioid receptor 1C and K_IR3.1 channel

The following sequences were used for μ-opioid receptor 1C cloning
First PCR,	Forward 5′-CGAGTCCGCAGCAAGCATTCAGAACCATGGAC-3′
	Reverse 5′-TCACCTGCCAAGCTGGCCTTCC-3′
Nested PCR,	Forward 5′-ATGGACAGCAGCGCCGGCCC-3′
	Reverse 5′-TCACCTGCCAAGCTGGCCTTCCCCGGATTCCTG-3′
For the HA tag insertion in μ-opioid receptor 1C
First PCR,	Forward 5′-CATACGATGTTCCAGATTACGCTGACAGCAGCGCCGGCCCAG-3′
	Reverse 5′-TCACCTGCCAAGCTGGCCTTCCCCGGATTCCTG-3′
Second PCR	Forward 5′-TTAAAAAAGCTTcaccATGTACCCATACGATGTTCCAGATTACGCTG-3′
	Reverse 5′-TTAAAAGAATTCACCTGCCAAGCTGGCCTTC-3′
The following sequences were used for KIR3.1 cloning
	Forward 5′-GTATTATGTCTGCACTCCGAAGGA-3′
	Reverse 5′-TTTTGCTATGTGAAACGGTCAGAGTT-3′

As for μ-opioid receptor 1C, the entire ORF was amplified by the primer sets of first PCR and nested PCRs. After being ligated to pCR 2.1 TOPO vector, HA tag sequence was inserted between the first and the second codon of the μ-opioid receptor 1C. In this case, for the first PCR, 3′ sequence of the HA tag (underline) was inserted before the second codon (bold). For the second PCR, restriction enzyme (Hind III) (italic) and kozac (lower case) sequences were added before the first codon (bold). In addition, the 5′ sequence of the HA tag (underline) was added after the first codon. EcoR I (italic) was added at the 3′ end of the ORF.

HA, haemagglutinin.