Full length human TR1 was amplified from pCMV6-XL4 vector by PCR using specific forward (TR1: 5'-GAAAGTCGAGGAGACAGTTAAGCATG-3') and reverse (TR1: 5'-CACAAGGAAAGGTCATGCTAAAACTG-3') primers and subsequently cloned into pcDNA 3.1 mammalian expression vector (Promega, Madison WI). Full length cDNA for human TR2 was recovered from pCMV6-XL4 by digestion with XbaI and EcoRI, purified by gel electrophoresis, and ligated into linear pcDNA 3.1 using T4 DNA Ligase. Insertion of wild type Sec-TR1 and Sec-TR2 full-length cDNAs, including the 3' SECIS elements, into pcDNA 3.1 was confirmed by sequencing using the appropriate forward and reverse primer sets (T7 Forward: BGH Reverse). PCR-based mutagenesis was performed by Mutagenex (Piscataway, NJ) to replace the Sec codon with Cys and was confirmed by sequencing. Expression vectors for human TRX1 and TRX 2 were purchased from Open Biosystems (Thermo Scientific, Waltham, MA). Expression vectors for cytosolic and mitochondrial targeted roGFPs were a kind gift from J.A. Melendez (College of Nanoscale Science and Engineering, Albany, NY).
Pcdna 3.1 mammalian expression vector
Manufactured by Promega
The pCDNA 3.1 mammalian expression vector is a plasmid designed for high-level, transient expression of recombinant proteins in mammalian cell lines. It contains a cytomegalovirus (CMV) promoter for efficient transcription initiation and the SV40 origin of replication for episomal replication in cells expressing the SV40 large T antigen.
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Lab products found in correlation
2 protocols using pcdna 3.1 mammalian expression vector
1
Cloning and Expression of Selenoproteins
2
Recombinant SARS-CoV-2 3CLpro Expression
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Standard PCR was used to amplify the two GFP fragments, β-strand 1–9 and β-strand 10–11. The ten amino acids linker and cleavage sites of 3CLpro and E5/K5 amino acid sequences were inserted into the GFP construct using overlap-extension PCR, as we described previously [26 (link)]. The purified DNA of GFP constructs and 3CLpro (GenBank code: MN908947.3, ORF1ab polyprotein residues 3264–3569,) were sequenced for any mutations, and the intact DNA fragments were cloned in pGEM®-T cloning vector (Cat.# A1360, Promega), amplified in the E. coli, retrieved and sub-cloned in pcDNA3.1 mammalian expression vector (Cat.# V79020, ThermoFisher). The HEK293T cells (ATCC) were co-transfected with the recombinant pcDNA3.1 plasmids using Lipofectamine 2000 (Cat.# 11668-019, Invitrogen).
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