A cDNA construct with a triple FLAG tag fused to the N‐terminus of CLN7 was generated by amplifying human MFSD8 cDNA (NM_152778, RZPD clone IRATp970E0532D6; imaGenes, Berlin, Germany) using primers 3xFLAG pCMV10‐CLN7 F/R and cloning this into the p3xFLAG‐CMV10 vector (Sigma‐Aldrich, St. Louis, Missouri). The cDNA coding for human mutant CLN7 Ile67Glufs*3 was purchased (Source BioScience, Nottingham, UK) and amplified. Primers can be found in Table S6 . PCR products were separated and purified from agarose gels, cleaved with restriction enzymes and cloned into the p3xFLAG‐CMV10 expression vector (Sigma‐Aldrich, St. Louis, Missouri) generating the mutant 3xFLAG p.Ile67Glufs*3.
P3xflag cmv 10 vector
Manufactured by Merck Group
Sourced in United States
The P3XFLAG-CMV-10 vector is a laboratory tool used for protein expression and purification. It is designed to add a 3xFLAG epitope tag to the target protein, allowing for efficient detection and purification of the expressed protein.
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Lab products found in correlation
Puromycin, by Thermo Fisher Scientific (5 mentions)
Neomycin, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Plko 1 puro, by Merck Group (2 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (2 mentions)
P3xflag cmv 10 expression vector, by Merck Group (1 mentions)
Prescission protease, by Merck Group (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
Pcdna3 vector, by Thermo Fisher Scientific (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Myc hulk1, by Addgene (1 mentions)
Cdna synthesis kit, by Toyobo (1 mentions)
Polyethyleneimine, by Merck Group (1 mentions)
Riboex, by GeneAll (1 mentions)
Circrna mini vector, by Addgene (1 mentions)
Prset b vector, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Plasmid mini kit, by Qiagen (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
31 protocols using p3xflag cmv 10 vector
1
Generating FLAG-tagged Mutant CLN7 Construct
2
Purification and Characterization of MPP1 Mutants
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All the sequences of primers for the construction of plasmids used in this study are summarized in the Supplementary Table 1 . The MPP1-truncated mutants (MPP1-Mut1-5) were subcloned into the pGEX-6p1 vector and expressed as soluble GST tagged proteins in Escherichia coli BL21 (DE3) or LEMO (DE) cells. Full length MPP1-GST and its truncated mutants were isolated in native conditions based on HBS buffers (10 mM HEPES, 150 mM NaCl, pH 7,4) and immobilized on glutathione-Sepharose 4B beads (GE Healthcare). The GST tag was cleaved off on the column with the PreScission protease (Sigma) according to the manufacturer's protocol. Purified recombinant proteins were validated by SDS-PAGE and Coomassie Blue staining (see Fig. S2 ). His-tagged flotillin 1 and 2 were purified under denaturation conditions as described previously23 (link). After purification recombinant proteins were dialyzed into HBS-T (HBS-0.05% Tween-20) buffer and subsequently used for SPR binding study. For mammalian cell experiments the MPP1-Mut4-FLAG construct was additionally cloned into the p3XFLAG-CMV-10 vector (Sigma, St. Louis, MO).
3
Cloning of HPV Oncogenic Genes
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The HPV16 and HPV18 E6 open reading frames were cloned into the p3XFLAG-CMV10 vector (Sigma-Aldrich, St. Louis, MO, USA) by standard PCR techniques. p3XFLAG-CMV10 vector harbours an ampicillin resistance cassette and a CMV promoter. HPV18 E7 and E1 sequences were cloned into the pcDNA3 vector (Invitrogen) adding at the N-terminus a 5X-histidine tag and a HA tag, respectively. HPV18 E2 expressing plasmid was generated by cloning the E2 coding region into the pcDNA3 vector. pcDNA3 vector harbours an ampicillin resistance cassette and a CMV promoter. Identity of each plasmid was verified by automated sequencing. HA tagged HPV16 E7 expressing plasmid was a kind gift from Dr. Lawrence Banks (ICGEB, Italy).
4
Generating Stable Cell Lines
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To generate stable overexpressing cell lines, H1299 or HCT116 cells were transfected with the p3XFLAG-CMV10 vector (Sigma-Aldrich) containing full-length human NRF3 or GFP. The transfected cells were selected with G-418. To generate stable knockdown cell lines, HCT116 or HCT116 p53KO cells were transfected with the piGENE hU6 plasmid (iGENE Therapeutics) containing human NRF3 target or control sequences (see Table S3 in the supplemental material). The transfected cells were selected with puromycin.
5
Generation of Hath1 Overexpression Construct
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The generation of the 4ACAT stabilized β-catenin construct, where the residues S33, S37, T41 and S45 were mutated to Alanine and the MUC4 promoter construct has been described in a previous publication [35 (link)]. For the Hath1 overexpression construct, the Hath1 cDNA was amplified from the HCT116 cell line using the following primers: HindIII-Hath1-FP: 5′-CATAAATAAGCTTTC CCGCCTGCTGCATGCAGAAG-3′and BamHI-Hath1- RP:5′-CTACAATGGATCCCTAACTTGCCTCATCCG AGTCAC-3′. The amplified cDNA was then ligated into the PCR2.1 vector using TOPO-TA cloning kit (Thermo Fisher Scientific #K451020). Following a restriction digestion with HindIII and BamH1, the released insert cloned into the HindIII and BamHI (New England Biolabs, #R0104Sand #R0136S) digested p3XFLAG- CMV-10 vector (Sigma-Aldrich, # E7658) and sequenced at the UNMC sequencing core facility.
6
Plasmid construction and mammalian expression
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The 3xFlag-hGABARAPL1 plasmid was generated by subcloning the PCR-amplified human GABARAPL1 cDNA into a p3xFLAG-CMV 10 vector (Sigma). HA-MAP1LC3A, HA-MAP1LC3B, HA-MAP1LC3C, HA-GABARAPL1, HA-GABARAP, HA-GABARAPL2 and Myc-hULK1 were purchased from Addgene (plasmid #137756, #137757, #137758, #137759, #137760, #137761, and #31961, respectively)51 (link), 52 (link). Flag-hp62 was kindly provided by Dr. Masaaki Komatsu53 (link).
7
Cloning and Expression of SLC3A2 and HCV Proteins
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Total cellular RNAs were isolated from Huh7 cells by using RiboEx (GeneAll), and full-length SLC3A2 was amplified by a primer set (sense, 5′-AAG CGG CCG CTA TGG AGC TAC AGC CT-3′; antisense, 5′-AAG GTA CCT CAG GCC GCG TAG GGG AA-3′) from cDNA synthesized by using a cDNA synthesis kit (Toyobo) according to the manufacturer’s instructions. PCR products were inserted into the BamH1 and Xba1 sites of plasmid pEF-V5-His (Invitrogen) to generate V5-tagged SLC3A2, and the Not1 and Kpn1 sites of the p3xFlag-CMV10 vector (Sigma-Aldrich) to generate Flag-tagged SLC3A2 expression plasmid. Myc-tagged HCV core, NS3, NS4B, NS5A, and NS5B, V5-tagged HCV E2, E1E2, NS3/4A plasmids were described previously12 (link),39 (link). All DNA transfections were performed by using polyethyleneimine (Sigma-Aldrich) as we described previously12 (link).
8
Overexpression of CMTr1 and DHX15
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The full-length cDNAs of CMTr1 and DHX15 were obtained from Source BioScience and subcloned into p3xFLAG-CMV®-10 vector (Sigma), introducing an N-terminal FLAG-tag, optionally cleavable with PreScission protease, for overexpression in HEK293 cells [10 (link)]. For overexpression in bacteria, DHX15 was cloned into the pET28 vector, introducing a C-terminal His-tag. Variants of CMTr1 were constructed using polymerase chain reaction (PCR) with p3xFLAG-CMV10_CMTr1 as a template. DNA construct for expression of the deletion variant that contained the N-terminal part of CMTr1, with the NLS and G-patch domain, was prepared by inserting a stop codon after the triplet coding for Arg133 (forward primer (fv) 5′-TGACAGGAGCTGAACGTGGACTG-3′, reverse primer (rv) 5′-TCACCGGAGTGTCAGACCCAAG-3′). The variant without the G-patch domain was prepared by removing a region that encodes residues 1–135 (fv 5′-GACCAGGAGCTGAACGTGG-3′, rv 5′-GGCGGCCGCAAGCTTGTC-3′).
9
Cloning and Characterization of Plasmids
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The expression plasmids used in the study were generated according to standard molecular biology techniques. All the plasmids were subjected to sequencing verification. Insig-1 was cloned into p3Xflag-CMV-10 vector (Sigma-Aldrich) by a PCR-based method. The following plasmids were described as previously reported: Myc-tagged PAQR3, green fluorescent protein (GFP)-fused PAQR3 and short hairpin RNA (shRNA) targeted for human PAQR3 (refs 17 (link), 40 (link)); Flag-tagged SREBP-2, Flag-tagged SREBP-1a and the SRE-luciferase reporter34 (link).
10
Molecular Cloning and Transfection Techniques
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Human cDNAs were synthesized using TSINGKE (Wuhan, China) to construct circCRIM1, IGF2BP1, and HLA-F overexpression plasmids. The circCRIM1 was cloned into the pcDNA3.1(+) CircRNA Mini Vector (addgene #60648); in addition, HLA-F and full-length or truncated proteins of IGF2BP1 (Supplementary Table S1 ) were cloned into the p3XFLAG-CMV-10 vector (Sigma-Aldrich). Specific short hairpin RNAs (shRNAs) for circCRIM1 were cloned into pLKO.1-puro (Sigma-Aldrich). Transfection was performed using Lipofectamine 3000 (Life Technologies) according to the manufacturer’s instructions. Neomycin or puromycin (Invitrogen) was used to screen stably transfected cell lines. An empty vector was applied as a control (Supplementary Table S1 ).
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