Constructs for 2x-myc- and 3x-flag- tagged LRRK2 full length and domains, GAK, BAG5, RAB29 and GUS have been described previously (Beilina et al., 2014 (link); Taymans et al., 2011 (link)). VPS52 full length and domain constructs were PCR amplified with the following primers: VPS52 full length: F_flVPS52 and R_flVPS52; ΔC-VPS52: F_ΔcVPS52 and R_ΔcVPS52; ΔN -VPS52: F_ΔnVPS52 and R_-ΔnVPS52; ΔC/ΔN-VPS52(Sac2): F_SacVPS52 and R_SacVPS52 and cloned into pCR8/GW/TOPO vector (Thermo Scientific). Full-length VPS52 and domains were transferred into the pCMV-2xmyc-DEST, pDEST53-DEST, pAcGFP-DEST and p3xflag-HD-DEST vectors using Gateway recombination technology (Thermo Scientific). The generation of a plasmid encoding Syndetin-GFP was previously described. A plasmid encoding VPS54-GFP was generated by using Homo sapiens VPS54 cDNA (Origene, MD) subcloned into pEGFP-N1 (Clontech) vector by Gibson assembly using F-VPS54 and R-VPS54; F_vector and R_vector primers.
Pcmv 2xmyc dest
Manufactured by Thermo Fisher Scientific
The PCMV-2xmyc-DEST is a vector designed for the expression of recombinant proteins in mammalian cells. It contains a cytomegalovirus (CMV) promoter for high-level expression, a N-terminal 2xmyc tag for detection and purification, and the Gateway cloning system for convenient insertion of genes of interest.
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2 protocols using pcmv 2xmyc dest
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Cloning and Expression of VPS52 Constructs
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Cloning and Expression of VPS52 Constructs
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Constructs for 2x-myc- and 3x-flag- tagged LRRK2 full length and domains, GAK, BAG5, RAB29 and GUS have been described previously (Beilina et al., 2014 (link); Taymans et al., 2011 (link)). VPS52 full length and domain constructs were PCR amplified with the following primers: VPS52 full length: F_flVPS52 and R_flVPS52; ΔC-VPS52: F_ΔcVPS52 and R_ΔcVPS52; ΔN -VPS52: F_ΔnVPS52 and R_-ΔnVPS52; ΔC/ΔN-VPS52(Sac2): F_SacVPS52 and R_SacVPS52 and cloned into pCR8/GW/TOPO vector (Thermo Scientific). Full-length VPS52 and domains were transferred into the pCMV-2xmyc-DEST, pDEST53-DEST, pAcGFP-DEST and p3xflag-HD-DEST vectors using Gateway recombination technology (Thermo Scientific). The generation of a plasmid encoding Syndetin-GFP was previously described. A plasmid encoding VPS54-GFP was generated by using Homo sapiens VPS54 cDNA (Origene, MD) subcloned into pEGFP-N1 (Clontech) vector by Gibson assembly using F-VPS54 and R-VPS54; F_vector and R_vector primers.
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