We used a TMEM163-mCherry expression construct [27 (link),28 (link)] to change a specific amino acid sequence corresponding to the non-synonymous TMEM163 SNPs used in the study (Supplemental Table S2 ). Using the online In-Fusion™ HD (IF) cloning system primer design website (Takara Bio), we made primer sets for TMEM163 SNP, ZIP1 (a kind gift of Dr. David J. Eide, University of Wisconsin), and the TMEM163 double aspartate to alanine substitution mutation located at positions 124 and 128 (D124A/D128A) (Supplemental Table S3 ). We used the human ZIP1 influx transporter as a treatment control since it has been reported to localize within the PM of cells [34 (link)], which is similar to the TMEM163 protein localization [28 (link)]. Note that the double aspartate to alanine mutations were previously reported to decrease zinc binding and inactivate protein function in rodent Tmem163 at positions 123 and 127 (Tmem163-D123A/D127A) [31 (link)]. Following SDM of the D124A/D128A and SNP constructs, we verified the DNA sequence integrity of all expression vectors by commercial sequencing (Retrogen, San Diego, CA) and analyzed the clone sequences using Lasergene SeqMan v. 15 (DNAStar, Madison, WI).
In fusion hd if cloning system primer design website
Manufactured by Takara Bio
The In-Fusion™ HD (IF) cloning system primer design website provides a tool for users to design primers for the In-Fusion HD cloning method. The website allows users to input their desired sequences and generates the appropriate primer sequences for the cloning process.
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2 protocols using in fusion hd if cloning system primer design website
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TMEM163 Mutant Constructs Generation
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TMEM163 Mutant Constructs Generation
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We used a TMEM163-mCherry expression construct [27 (link),28 (link)] to change a specific amino acid sequence corresponding to the non-synonymous TMEM163 SNPs used in the study (Supplemental Table S2 ). Using the online In-Fusion™ HD (IF) cloning system primer design website (Takara Bio), we made primer sets for TMEM163 SNP, ZIP1 (a kind gift of Dr. David J. Eide, University of Wisconsin), and the TMEM163 double aspartate to alanine substitution mutation located at positions 124 and 128 (D124A/D128A) (Supplemental Table S3 ). We used the human ZIP1 influx transporter as a treatment control since it has been reported to localize within the PM of cells [34 (link)], which is similar to the TMEM163 protein localization [28 (link)]. Note that the double aspartate to alanine mutations were previously reported to decrease zinc binding and inactivate protein function in rodent Tmem163 at positions 123 and 127 (Tmem163-D123A/D127A) [31 (link)]. Following SDM of the D124A/D128A and SNP constructs, we verified the DNA sequence integrity of all expression vectors by commercial sequencing (Retrogen, San Diego, CA) and analyzed the clone sequences using Lasergene SeqMan v. 15 (DNAStar, Madison, WI).
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