Pcmv 3tag 6

Manufactured by Agilent Technologies

Sourced in United States

The PCMV-3Tag-6 is a plasmid vector designed for the expression of recombinant proteins in mammalian cell lines. It features a CMV promoter for high-level expression, a 3x FLAG tag, and a 6x His tag for convenient detection and purification of the target protein.

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Lab products found in correlation

Pcmv 3tag 7, by Agilent Technologies (2 mentions) Pgex 6p 1, by GE Healthcare (2 mentions) Pcmv myc, by Takara Bio (2 mentions) Human multiple tissue cdna panel, by BD (2 mentions) Pcr purification kit, by Tiangen Biotech (1 mentions) Flag traf6 21624, by Addgene (1 mentions) Flag becn1, by Agilent Technologies (1 mentions) Pegfp c3, by Takara Bio (1 mentions) Pcmv 3tag 6, by Addgene (1 mentions)

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PCMV-3Tag-6 vector (5 citations)

6 protocols using pcmv 3tag 6

Generation of FLAG-AQP0 and Myc-AQP0 Fusion Proteins

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To create FLAG-AQP0 and Myc-AQP0 fusion proteins, the PCR products of WT-AQP0 and Y219*-AQP0 were digested with BamHI and XhoI restriction enzymes, respectively, purified with a PCR purification kit (Tiangen Biotech, Beijing, China), and subsequently cloned into the digested mammalian expression vector, pCMV-3Tag-6(Agilent Technologies, Shanghai, China), which contained an N-terminal triple FLAG epitope tag;and pCMV-3Tag-7 (Agilent Technologies, Shanghai, China), which contained an N-terminal tripleMyc epitope tag. All of the constructs were verified by direct sequencing.

Song Z., Wang L., Liu Y, & Xiao W. (2015). A Novel Nonsense Mutation in the MIP Gene Linked to Congenital Posterior Polar Cataracts in a Chinese Family. PLoS ONE, 10(3), e0119296.

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Plasmid Construction and Modification

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Flag-TRAF6 (21624) and Flag-BECN1 (24388) plasmids were purchased from Addgene (Cambridge, MA 02142, USA). HA-tagged Ub plasmids were obtained from Dr. J. H. Shim (University of Massachusetts Medical School, USA). Using the Flag-TRAF6 or Flag-BECN1 plasmid as templates, full-length TRAF6 and BECN1 were cloned into pCMV-3Tag-7 (Agilent technologies, 240202) to generate Myc-TRAF6 and Myc-BECN1, respectively. Beta Arrestin 2/ARRB2 cDNA ORF Clone (HG15078-CF) was purchased from Sino Biological US Inc. (Wayne, PA, USA). ARRB2 full length was cloned into a pCMV-3Tag 6 vector (Agilent technologies, 240200) to generate Flag-ARRB2. Truncated mutants of Flag-TRAF6 and Flag-BECN1 were generated as previously described [25 (link), 26 (link)].

Kim J.Y., Shin J.H., Kim M.J., Kang Y., Lee J.S., Son J., Jeong S.K., Kim D., Kim D.H., Chun E, & Lee K.Y. (2023). β‐arrestin 2 negatively regulates lung cancer progression by inhibiting the TRAF6 signaling axis for NF-κB activation and autophagy induced by TLR3 and TLR4. Cell Death & Disease, 14(7), 422.

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Cloning and Mutagenesis of OTUD4 Isoforms

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Human OTUD4 cDNA coding for isoform 4 (1114 aa) was cloned in pEGFP-C3 (Clontech), pCMV-3Tag-6 (Agilent Technologies), pN3HA (kindly provided by Christoph Thiele, University of Bonn, Germany) or pGEX-6P-1 (GE Healthcare). The catalytically inactive mutant OTUD4 C45A was produced by site-directed mutagenesis (SDM). siRNA-resistant OTUD4 constructs were made by introducing four silent mismatches (bold) in the siRNA targeting region by using the following primer sequence (fwd) for SDM: 5′-GGGAACCAAATGTCTCCCCATCACAGGTAACAGAAAATAATTTTC-3′.
GFP-SMN1 was from Addgene, deposited by Greg Matera (Addgene #37057, Shpargel and Matera, 2005 (link)), pFRT-TODestFLAGHA_HuB (Addgene #65755) and pDESTmycIGF2BP3 (Addgene #19879; Landthaler et al., 2008 (link)), deposited by Thomas Tuschl.
SMN1 was subcloned into pmNeonGreen-C1 (Gentaur Europe BVBA). The resulting mNeonGreen–SMN1 cassette was then excised and subcloned into a modified p-βactin vector (Kapitein et al., 2010 (link)), kindly provided by Casper Hoogenraad (Utrecht University, The Netherlands). For live-cell imaging, OTUD4 was cloned into p-βactin-mOrange2-C1 (mOrange2 was amplified from mOrange2–EB3-7, Addgene #57953, a deposited by Michael Davidson).

Das R., Schwintzer L., Vinopal S., Aguado Roca E., Sylvester M., Oprisoreanu A.M., Schoch S., Bradke F, & Broemer M. (2019). New roles for the de-ubiquitylating enzyme OTUD4 in an RNA–protein network and RNA granules. Journal of Cell Science, 132(12), jcs229252.

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Stable Expression and Knockdown of TRIM16

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To stably express TRIM16, the full-length open reading frame (ORF) of TRIM16 (GenBank NM_001348120) was amplified from a Human Multiple Tissue cDNA Panel (BD Biosciences) and then cloned into pLVX-puro at the XhoI and EcoRI sites. To knock down TRIM16, shRNA targeting TRIM16 and a scramble sequence were synthesized by Invitrogen (Beijing, China). The sequence of shRNA against TRIM16 and a negative control were then inserted into the pLKO.1 vector and named sh-TRIM16 and sh-NC, respectively. The transcripts of CHIP (GenBank NM_005861) and RUNX2 (GenBank NM_001024630) were amplified by PCR and subcloned into the pCMV-3Tag6 (Agilent Technologies, Santa Clara, CA, United States) and pCMV-Myc (Clontech, Mountain View, CA, United States) vectors, respectively. The constructs were named Flag-CHIP or Myc-RUNX2. All constructs were confirmed by direct sequencing (Table 1). The pRK5-HA-Ubiquitin-WT and K48, K63 were gifts from Ted Dawson (Addgene, Watertown, MA, United States; plasmid # 17608; 17605, 17606).

Zhao Y., Zhai Q., Liu H., Xi X., Chen S, & Liu D. (2021). TRIM16 Promotes Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Modulating CHIP-Mediated Degradation of RUNX2. Frontiers in Cell and Developmental Biology, 8, 625105.

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Cloning and Mutagenesis of TRIAD3 Proteins

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Expression constructs for human TRIAD3B (NCBI accession number NM_207111.3) were cloned into a modified pcDNA3 vector coding for a C-terminal V5/His₆-tag (kindly provided by Tencho Tenev) or pN3HA vector with 3X N-terminal HA-tag (kindly provided by Christoph Thiele). cDNA coding for TRIAD3A was cloned into pCMV-3Tag-6 (Agilent) or pGEX-6P-1 (GE Healthcare). Constructs coding for TRIAD3B C-terminus (aa 562-923) or TRIAD3B-RBR fragments (aa 562-825/-835/-845) were cloned into pGEX-6P-1 (). Catalytically inactive point mutant TRIAD3B C745A (TRIAD3A C688A) and disease point mutants TRIAD3B G456E, Y539C, R717C and R751C were produced by site-directed mutagenesis (SDM).
cDNA coding for human Ankrd13D (IMAGE ID 5210878) was cloned into pCMV-3Tag-6. psigma2-EGFP was a gift from Tom Kirchhausen (Addgene plasmid # 53610) and was used as template to clone rat AP2s1 into pCMV-3Tag-6. Human ARC cDNA (IMAGE ID 4555433) was cloned into pN3HA. pCS2 HRS-RFP was a gift from Edward De Robertis (Addgene plasmid # 29685) and expression constructs for HRS and UIM deletion mutant HRS ΔUIM (Δ aa 258–277) were cloned into pCMV-3Tag-6. Mutant UIM construct HRS UIM* (L269A, S270A) was produced by SDM. Human VPS35 cDNA (IMAGE ID 30340379) was cloned into pCMV3Tag-6. pCW7 coding for His₆/c-myc-tagged yeast ubiquitin and ubiquitin mutant constructs K48R and K63R were a kind gift from Ron Kopito.

Schwintzer L., Aguado Roca E, & Broemer M. (2019). TRIAD3/RNF216 E3 ligase specifically synthesises K63-linked ubiquitin chains and is inactivated by mutations associated with Gordon Holmes syndrome. Cell Death Discovery, 5, 75.

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Cloning and Knockdown of TRIM16

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To stably express TRIM16, the full-length open reading frame (ORF) of TRIM16 (GenBank NM_001348120) was ampli ed from a Human Multiple Tissue cDNA Panel (BD Biosciences) and then cloned into pLVX-puro at the XhoI and EcoRI sites. To knock down TRIM16, siRNA targeting TRIM16 and a scramble sequence were synthesized by Invitrogen (Beijing, China). The sequence of shRNA against TRIM16 and a negative control were then inserted into the pLKO.1 vector and named sh-TRIM16 and sh-NC, respectively. The transcripts of CHIP (GenBank NM_005861) and RUNX2 (GenBank NM_001024630) were ampli ed by PCR and subcloned into the pCMV-3Tag6 (Agilent Technologies, Santa Clara, CA, USA) and pCMV-Myc (Clontech, Mountain View, CA, USA) vectors, respectively. The constructs were named Flag-CHIP or Myc-RUNX2. All constructs were con rmed by direct sequencing (Table 1). pRK5-HA-Ubiquitin-WT and K48, K63 were gifts from Ted Dawson (Addgene, Watertown, MA, USA; plasmid # 17608; 17605, 17606).

Zhao Y., Zhai Q., Liu H., Xi X., Chen D., & Liu D. (2020). TRIM16 Positively Regulates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Modulating the Ubiquitination and Degradation of RUNX2.

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