cDNA encoding the fluorescent protein cpVenus173 was amplified from the Gαi2-sensor v2.027 (link). The SNAPtag sequence was amplified using a SNAPtag-GABAB1 template kindly provided by J.P. Pin (Institut de Génomique Fonctionnelle, Montpellier, France)28 (link). Sequences encoding TagRFP (pTagRFP-C vector) and mCherry were purchased from Evrogen and Addgene, respectively. cDNA encoding HaloTag (pFC14K HaloTag® CMV Flexi® Vector) and NanoLuciferase (pFC32K Nluc CMV-neo Flexi® Vector) were purchased from Promega. Gαi2-FRET27 (link) sensor was kindly provided by J. Goedhart (Section Molecular Cytology, University of Amsterdam, Amsterdam, The Netherlands), the H187-EPAC-FRET sensor29 (link) was kindly provided by K. Jalink (The Netherlands Cancer Institute, Amsterdam, The Netherlands).
Pfc14k halotag cmv flexi vector
Manufactured by Promega
Sourced in United States
The PFC14K HaloTag CMV Flexi Vector is a plasmid vector that enables the expression and study of HaloTag-fusion proteins in mammalian cell lines. It provides a flexible cloning system and contains the CMV promoter for high-level expression of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Ptagrfp c vector, by Evrogen (1 mentions)
Pfc32k nluc cmv neo flexi vector, by Promega (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Pegfp n1 mammalian expression vector, by Takara Bio (1 mentions)
Halotag7, by Promega (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Pcdna3.1 mammalian expression vector, by Thermo Fisher Scientific (1 mentions)
4 protocols using pfc14k halotag cmv flexi vector
1
Fluorescent Protein and Tag Sensor Cloning
2
Myo19-3IQ-HaloTag Construct Generation
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The verified full-length human Myo19 cDNA12 (link) was used as a template to amplify Myo19-3IQ construct with primers 3 and 4 (Table S3 ) carrying AsiSI and Pmel restriction sites. After restriction, the fragment was inserted into SgfI and Eco53kI cut pFC14K HaloTag CMV Flexi Vector (Promega, Madison, WI, USA). Myo19-3IQ-HaloTag fragment was inserted into pF12A RM Flexi Vector (Promega), upgraded in-house with puromycin resistance gene to enhanced generation of stable cell lines according to manufacture protocol (Promega). pF12A RM Myo19-3IQ-HaloTag was generated by restriction ligation using AsiSI and SbfI where pFC14K Myo19-3IQ was a donor vector and pF12A RM was acceptor vector. The resulting plasmid (pF12A RM Myo19-3IQ-HaloTag) was then fully verified by sequence analysis. Human calmodulin light chain gene10 (link) was cloned to pF4A CMV Flexi Vector (Promega) according to the manufacturer’s protocol using primer 1 and 2 (Table S3 ). All PCR reaction were done with Phusion High fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and T4 PNK and ligase (NEB, Ipswich, MA, USA) were used according to the manufacturer’s instructions.
3
Cloning of Shootin1a, DCC, and Actin Constructs
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To generate pCMV-FLAG-shootin1a, the cDNA of human shootin1a was amplified by PCR with the primers Shootin1a F (5′-CCGCTCGAGATGAACAGCTCGGACGAGGAGAAG-3′) and shootin1a R (5′-CCGCTCGAGTTACTGGGAGGCCAGGATTCC CTTCAG-3′), and then subcloned into pCMV-FLAG vector (Toriyama et al., 2006 (link)). To generate pFC14K-DCC-Halotag, the cDNA of human DCC was amplified by PCR with the primers DCC F (5′-GAGGATCCATGGAGAATAGTCTTAGATG-3′), and DCC R (5′-CTGGATCCCTAAAAGGCTGAGCCTGTGATGG-3′), and then subcloned into pFC14K-Halotag CMV Flexi vector (Promega). To generate pCMV-EGFP-DCC ICD (intracellular domain), the cDNA of human DCC was amplified by PCR with the primers DCC ICD F (5′-GGTGAACTTCAAGATCCGCCACAA-3′), and DCC ICD R (5′-TGCAATAAACAAGTTAACAACAACAATTG-3′), and then subcloned into pCMV-EGFP vector (Agilent Technology). The preparation of pFN21A-HaloTag-actin has been described previously (Minegishi et al., 2018 (link)).
4
Protein fusion and expression techniques
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The HaloTag7 (Promega) coding sequence was amplified by PCR, and was fused at the C-terminus of mouse mGluR3 with an In-Fusion HD Cloning Kit (Clontech). To quantify the expression of wild type and HaloTag-fused mGluR3 by Western blotting analysis, the epitope sequence of the anti-bovine rhodopsin monoclonal antibody Rho1D4 was also fused at the C-terminus. The cDNAs of mGluR3s were introduced into the pcDNA3.1 mammalian expression vector (Invitrogen). The cDNAs of other GPCRs (ADRB2, HTR2A, HRH1, ADORA2A, FFAR4, CXCR4, F2R, GCGR) were purchased from Promega, and the receptor coding sequences were inserted into pFC14K HaloTag CMV Flexi Vector. The CD86 (M1-R277) coding sequence was amplified by PCR, and inserted into the pEGFP-N1 mammalian expression vector (Clontech), where the coding sequence of EGFP was swapped with that of HaloTag7. The SNAP-tag (NEB) coding sequence was amplified by PCR and inserted in the loop region between L91 and G92 of the mouse Gα o subunit coding cDNA, as reported previously (12) . The SNAP-tagged Gα o subunit coding sequence was inserted into the pFC15A HaloTag CMVd1 Flexi Vector without the HaloTag coding sequence to reduce the expression level for single-molecule imaging. The cDNA of GFP-tagged CLC was constructed as previously reported (45) (link).
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