cDNAs encoding full-length human ANO1 (GenBank Accession No. XM_011545125) and human 14-3-3γ (NM_012479.3) were obtained using a RT-PCR-based gateway cloning method (Invitrogen). Several mutants of ANO1 including ANO1(abc), ANO1(ac), ANO1(bc), ANO1(0), ANO1(abc)-Δa, ANO1 segment a (seg(a)), and ANO1 T9A were generated using full-length human ANO1 cDNA as a template via an EZchangeTM site-directed mutagenesis kit (Enzynomics). All constructs were subcloned into several vectors, including pDEST-FLAG-N, pDEST-HA-N, pDEST-GFP-N, and pDEST-mCherry-N, by gateway cloning (Invitrogen). The R18 peptide sequence (GVTQGALESTLDLEANMCL) was obtained using an EZchangeTM site-directed mutagenesis kit (Enzynomics).
Ezchange site directed mutagenesis kit
Manufactured by Enzynomics
Sourced in Cameroon
The EZchange site-directed mutagenesis kit is a tool designed for introducing specific mutations into DNA sequences. It provides a straightforward and efficient method for generating targeted genetic modifications.
Automatically generated - may contain errors
Lab products found in correlation
Psicheck 2 plasmid, by Promega (4 mentions)
Preceiver vector, by GeneCopoeia (3 mentions)
Ez fusion cloning kit, by Enzynomics (2 mentions)
Gateway cloning method, by Thermo Fisher Scientific (2 mentions)
Psicheck 2, by Promega (2 mentions)
Dual glo luciferase reporter assay system, by Promega (2 mentions)
Gateway cloning system, by Thermo Fisher Scientific (1 mentions)
Block it u6 rnai entry vector kit, by Thermo Fisher Scientific (1 mentions)
Gateway cloning, by Thermo Fisher Scientific (1 mentions)
Pcdna3 vector, by Thermo Fisher Scientific (1 mentions)
Pegfp n1 vector, by Takara Bio (1 mentions)
Pcep4 vector, by Thermo Fisher Scientific (1 mentions)
Gblocks gene fragment, by Integrated DNA Technologies (1 mentions)
Gateway lr cloning, by Thermo Fisher Scientific (1 mentions)
Multisite gateway lr recombination reaction, by Thermo Fisher Scientific (1 mentions)
Sypro orange dye, by Thermo Fisher Scientific (1 mentions)
P nitrophenyl butyrate p npb, by Merck Group (1 mentions)
Fugene hd, by Promega (1 mentions)
Pgl4 basic vector, by Promega (1 mentions)
T a vector, by RBC Bioscience (1 mentions)
54 protocols using ezchange site directed mutagenesis kit
1
Cloning and Mutagenesis of Human ANO1 and 14-3-3γ
2
Site-Directed Mutagenesis of pGL3-378
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Mutations with base substitution in the pGL3-378 construct were generated using a EZchangeTM Site-directed Mutagenesis kit (Enzynomics, Korea) according to the manufacturer’s protocol using oligonucleotide primers (Table 1 ). The desired mutations were verified by DNA sequencing.
3
Molecular Cloning and Mutagenesis of TREK Channels
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Full-length mouse TREK1 (GenBank accession number NM_010607), TREK2 (GenBank accession number NM_029911), mouse TRAAK (GenBank accession number NM_001403912), and mouse β-COP (GenBank accession number NM_033370) cDNA and the derived vectors were constructed using reverse transcription polymerase chain reac- tion (RT-PCR). TREK2ΔN, TREK2ΔC, TREK2ΔC1, TREK2ΔC2, TREK2ΔC3, TREK2ΔC4, and TREK2RRRAAA are mutants generated with the EZchangeTM site-directed mutagenesis kit (Enzynomics, Daejeon, Republic of Korea) using the mouse TREK2 cDNA as a template. Vectors pDEST-Flag-C and pDEST-EGFP-N were cloned using the Gateway cloning method. DsRed-Mem (Clontech Laboratories, Mountain view, CA, USA) was used to label the cell membranes, and pmScarlet_Giantin_C1 (Addgene, Teddington, UK) was used to label the Golgi apparatus.
4
Cloning and Mutagenesis of TTYH2 and β-COP
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To construct expression vectors for human TTYH2 (NM_032646.5) and human β-COP (NM_016451), full-length genes of both were amplified by an RT-PCR and cloned into several destination vectors (pDEST-GFP-N, pDEST-GFP-C, pDEST-HA-N, pDEST-HA-C, pDEST-mCherry-N, pDEST-GBK, and pDEST-GAD) by Gateway Cloning System (Invitrogen). TTYH2 related deletion mutants were obtained by full-length cDNAs as templates using the EZchangeTM Site-directed Mutagenesis Kit (Enzynomics). The shRNA vector for human TTYH2 was constructed using the BLOCK-iT U6 RNAi Entry vector kit (Invitrogen). The target sequence of TTYH2 shRNA was 5′-GGATTATCTGGACGCTCTTGC-3′ (CDS 1116–1136). The control Sc shRNA was constructed using the provided LacZ double-stranded control oligo.
5
Adenoviral Delivery of ANO1 and 14-3-3γ shRNAs
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The ANO1 and 14-3-3γ-shRNAs target sequences were; for ANO1 shRNA-1, 5′-GCATCCAGCTCAGCATCAT-3′ (1970–1990 nt); for ANO1 shRNA-2, 5′-CGTGTACAAAGGCCAAGTA-3′ (773–779 nt); for 14-3-3 γ-shRNA-1, 5′-ACGAGGACTCCTACAAGGAC-3′(635–654 nt); for 14-3-3 γ-shRNA-2, 5′-GATTAGGCCTGGCTCTTAACT-3′(515–535 nt). pSicoR-ANO1-shRNAs and pSicoR-14-3-3 γ-shRNAs were constructed using the EZchangeTM site-directed mutagenesis kit (Enzynomics) and confirmed by sequencing. For adenovirus production, For production of adenovirus, pSicoR cassette containing U6 promoter, shRNA sequences and CMV-mCherry flanked by loxP sites was transferred to pAd/CMV/V5-DESTTM vector using a gateway system and concentrated virus was produced by VirapowerTM Adenoviral Gateway Expression Kit (Invitrogen), according to the manufacturer’s instructions.
6
Generating PTPN2-MT Substrate Trapping Mutant
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The PTPN2-MT (D182A) is mutated at Asp 182 residue to Ala and has the similar affinity for substrate with wild-type, but the catalytic activity was reduced. Therefore, PTPN2-MT can form a stable complex with tyrosine phosphorylated substrates and protect those substrates from endogenous phosphatase-induced dephosphorylation [19 ]. The EZchangeTM site-directed mutagenesis kit was used to generate the PTPN2 substrate trapping D182A mutant (PTPN2-MT) in accordance with the manufacturer’s protocol (Enzynomics, Daejeon, Korea). The generated mutation of PTPN2 was validated with DNA sequencing.
7
Cav-2 Mutants and Photoactivation Constructs
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A full-length Cav-2 cDNA (GenBank accession no. NM_131914) was subcloned into pcDNA3 vector (Invitrogen) as described (36 (link),37 (link)). Cav-2 oligomerization domain deletion (Δ47–86-Cav-2) and tyrosine phosphorylation deficient (Y19A-Cav-2) mutants were generated by polymerase chain reaction (PCR) mutagenesis from the wild type (WT) pcDNA-Cav-2. The resulting entry vectors of WT and mutants were converted into self-constructed GFP tagging destination expression vector (pEGFP-N1 vector, Clontech Laboratories). The Cav-2 and Δ47–86-Cav-2 cloned into pEGFP-N1 vector were point-mutated at codon 203 from threonine to histidine in the GFP sequence of pEGFP-N1 vector as described (6 (link)) by PCR mutagenesis to generate Cav-2-PAGFP and Δ47–86-Cav-2-PAGFP for photoactivation. Cav-2 domain truncation mutants including Δ1–13-Cav-2, Δ1–46-Cav-2, Δ1–70-Cav-2, Δ47–86-Cav-2 and Δ120–162-Cav-2 were generated by using the WT Cav-2-GFP (residues 1–162) as template via EZchange site-directed mutagenesis kit (Enzynomics). The resulting entry vectors of WT and mutants were converted into self-constructed MBP tagging destination expression vector (pMGWA vector). All expression vectors were verified by sequencing.
8
Cloning and Mutating DKK1 3'-UTR
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The full-length 3'-UTR of DKK1 was amplified from the cDNA of AGS cells. The 3'-UTR of DKK1 was then cloned into XhoI/NotI sites located between the Renilla luciferase-coding sequence and the poly (A) site of the psiCHECK-2 plasmid (Promega, Madison, WI, USA) to produce psiC_DKK1. The primers used to amplify DKK1 were 5'-TCTAGGCGATCGCTCGAGACCAGCTATCCAAATGCAGT-3' and 5'-TTATTGCGGCCAGCGGCCGCAGGTATTATTAATTTATTGGAAACTATTTTTGA-3'. Mutations were introduced into the seed match sequence of psiC_DKK1 to produce psiC_DKK1m using an EZchange site-directed mutagenesis kit (Enzynomics, Daejeon, South Korea). The primers used for this purpose were 5'-ACAAAATTTTTGTACACATTGATTGTTATCTTGACTGA-3' and 5'-TATCAAGAGGAAAAATAGGCAGTGCAG-3'. The DKK1 coding sequences (DKK1- CDS) were amplified by using cDNA prepared from AGS cells. The primers used were 5'-TGCTAGCGGCCGCTCGAGATGATGGCTCTGGGCGCAGC-3' and 5'-CTTATCATGTCTGGATCCTTAGTGTCTCTGACAAGTGTG-3'. The amplicons were cloned into the XhoI/BamHI sites of the pCEP4 vector (Invitrogen) by using an EZ-Fusion cloning kit (Enzynomics, Daejeon, South Korea). The constructed DKK1 expression vector (pCEP4DKK1) contained a hygromycin selection marker for enrichment of transfected cells.
9
Cloning and Manipulation of Ion Channel Genes
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cDNAs encoding full-length mouse TWIK-1 (NM_008430) and mouse TASK-3 (NM_001033876) were obtained by using the Gateway cloning method (Invitrogen, Carlsbad, CA, USA). cDNAs encoding full-length human NTSR-1 (NM_002531) and human NTSR-2 (NM_012344) were synthesized by designing gene blocks (gBlocks® Gene Fragments; Integrated DNA Technologies, Coralville, IA, USA) and constructing entry clones using the Gateway BP cloning method (Invitrogen). The constructs were cloned into several vectors, including pDEST-HA-N, pDEST-FLAG-N, pDEST-IRES2-GFP, and pDEST-IRES2-mCherry by using Gateway LR cloning (Invitrogen). To construct the concatenated TWIK-1 and TASK-3, TASK-3 and TWIK-1 were recloned into the pDONR207 P1P5R and pDONR207 P5P2 vectors, respectively, via two independent BP reactions (Invitrogen), followed by a MultiSite Gateway LR recombination reaction (Invitrogen) according to the manufacturer’s guidelines to produce pDEST-IRES2-GFP. The target regions of the shRNA (mouse TWIK-1: 5′-GCATCATCTACTCTGTCATCG-3′; mouse TASK-3: 5′-GCTGGTGTCCAGTGGAAATTC-3′) were obtained by oligonucleotide-directed mutagenesis using an EZchange site-directed mutagenesis kit (Enzynomics, Daejeon, Korea).
10
Site-Directed Mutagenesis of CYP1A1 Variants
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Site-directed mutagenesis was conducted to generate various single residue variants of
dog and human CYP1A1 using an EZchange site-directed mutagenesis kit (Enzynomics)
according to the manufacturer’s instructions. Primers used for mutagenesis are listed in
Supplementary Table 1 .
dog and human CYP1A1 using an EZchange site-directed mutagenesis kit (Enzynomics)
according to the manufacturer’s instructions. Primers used for mutagenesis are listed in
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