The mGluR1 Lc fragment was digested from pCAGGS-mGluR1Lc construct (a gift from Dr. Michisuke Yuzaki) and subcloned into the pUAST vector.
We prepared a reference construct by digesting the HSV-TK promoter, between the BamH1 site and Hind III sites, from the pRL-TK vector (Promega Corporation, Madison, WI, USA) that expresses Renilla luciferase, and cloning this fragment into the pGL4.18 [ luc2p/Neo] vector (Promega) that expresses Firefly luciferase.
A series of experiments was constructed to digest the monazite sample with a low amount of concentrated H2SO4 and to explore the separation possibility of Th from REEs in successive steps at different BaCl2 weights that act as precipitating agent.
We generated a polyA-TRE construct by digesting pREV-TRE with XhoI and pSV40-ZEO2 (Invitrogen Corporation) with EcoRI.
This construct was digested with EcoRV, producing a blunt-ended vector into which the Gateway Reading Frame A (a 1.6-kb fragment containing the chloramphenicol-resistant gene and the ccdB gene flanked by attR sites) was cloned according to manufacturer's protocol (Invitrogen).
The resulting construct was digested by NotI and a fragment containing the amplified sequence of HvCEBiP was introduced into the NotI site of pSL038-1 in the antisense orientation.
After ligation of the GEX adapter 1, the construct was digested with MmeI to create a 21 base pair fragment downstream of the GEX adapter 1.
The resulting construct was digested with NotI and AflII to insert a polyadenylation addition sequence, which had been excised from pEGFP-1 (Clontech) using NotI and AflII.
The CLCN4 pcDNA 3.1 construct was digested with NsiI and BamHI to generate a 5′ fragment of the cDNA sequence.
The construct was digested with BsaI and AatII and ligated with a 589 bp siaA fragment (nt. 571 to 1160), amplified using primers NG1306 (5'-GCAGACGTCGCCTAGTGCAAATGGGAGAA-3') and NG1307 (5'-gcaggtctcatctgtgctggtgcgagtatc).
The DNA sequence encoding each final SELP construct was digested with Eam 1104I, blunt-ended and subcloned into pCM13 (a modified pET-25 expression vector).
