Pcdna3.1 c k dyk

Manufactured by GenScript

Sourced in United States

PcDNA3.1+/C-(K)-DYK is a mammalian expression vector that can be used for the overexpression of proteins in cell culture systems. It contains a strong cytomegalovirus (CMV) promoter for efficient transcription and a C-terminal DYK (Asp-Tyr-Lys) tag for detection and purification purposes.

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Lab products found in correlation

Q5 site directed mutagenesis kit, by New England Biolabs (2 mentions) Brilliant 3 ultra fast sybr green qrt pcr master mix, by Agilent Technologies (1 mentions) Neon kit, by Thermo Fisher Scientific (1 mentions) Sybr green primers, by Merck Group (1 mentions) Puromycin, by Addgene (1 mentions) Mission sirna, by Merck Group (1 mentions) Rnaimax, by Thermo Fisher Scientific (1 mentions) Fugene hd, by Promega (1 mentions) Dpni enzyme, by New England Biolabs (1 mentions) Anti flag m2 antibody, by Merck Group (1 mentions) G418 antibiotic, by Thermo Fisher Scientific (1 mentions) Scc049, by Merck Group (1 mentions) Endofectin max transfection reagent, by GeneCopoeia (1 mentions) Pfu turbo dna polymerase, by Agilent Technologies (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Ac16 human cardiomyocyte cell line, by Merck Group (1 mentions) Nucleofector sg kit, by Lonza (1 mentions) Nucleofector sf kit, by Lonza (1 mentions) Dual luciferase reporter assay, by Promega (1 mentions) Amaxa nucleofector, by Lonza (1 mentions)

21 protocols using pcdna3.1 c k dyk

Overexpression of key oncogenes in esophageal cells

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The vectors pCMVHA E2F1^{66 (link)} (Item ID 24225, Addgene), pLX_TRC317 (TRCN0000481188, Sigma-Aldrich) and pcDNA3.1 + /C-(K)-DYK (Clone ID: OHu19407D, Genscript) were used to induce E2F1, MCM7, and PAK1 overexpression, respectively. FLO-1 cells were transfected according to the manufacturer’s protocol, while CP-A cells were nucleofected following the Neon™ kit protocol (Thermo Fisher), with 2 pulses of 1200 V for 20 ms. Overexpressing cells were selected with either G481/Geneticin (E2F1, PAK1) or Puromycin (MCM7). Empty vectors carrying G418 (pcDNA3.1 + /C-(K)-DYK, Genscript) or Puromycin (Item ID 85966, Addgene) resistance were used as controls. The RNA from transfected cells was used to assess gene overexpression via quantitative RT-PCR using predesigned SYBR green primers (Sigma-Aldrich; Supplementary Table 3) and Brilliant III Ultra-Fast SYBR Green QRT-PCR Master Mix (Agilent Technologies). The average expression level across triplicates (e) was relativised to the average expression level of β-2-microglobulin (c):

r = e - c

where r is the relative gene expression. The fold change (fc) between the relative gene expression after overexpression and the relative gene expression in the control condition (r_c) was calculated as:

f c = 2^{(r_{c} - r K D)}

Each sample was assessed in triplicate and each experiment was repeated in biological duplicate.

Mourikis T.P., Benedetti L., Foxall E., Temelkovski D., Nulsen J., Perner J., Cereda M., Lagergren J., Howell M., Yau C., Fitzgerald R.C., Scaffidi P, & Ciccarelli F.D. (2019). Patient-specific cancer genes contribute to recurrently perturbed pathways and establish therapeutic vulnerabilities in esophageal adenocarcinoma. Nature Communications, 10, 3101.

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Optogenetic Modulation of Synaptotagmin 1

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pAAV.CAG.hChR2(H134R)-mCherry.WPRE.SV40 and the AAV9 viral particles produced from the plasmid were a gift from Karl Deisseroth (Addgene viral prep #100,054-AAV9). Syt1 shRNA target sequence 5’-GAGCAAATCCAGAAAGTGCAA-3’ was previously determined and validated^{17 (link)}. pAAV.H1.Syt1shRNA.CAG.hChR2(H134R)-mCherry was constructed and packaged by VectorBuilder by modifying the original hChR2 pAAV plasmid to include the Syt1 shRNA behind an H1 promoter (Vector ID VB170324-1065bbv).
pAAV.H1.scrambledshRNA.CAG.hChR2(H134R)-mCherry was also constructed and packaged by VectorBuilder by modifying the Syt1 shRNA AAV plasmid to instead express a scrambled shRNA (Vector ID VB191113-1716anp). Rat synaptotagmin 1 in pcDNA3.1⁺/C-(K)DYK was acquired from GenScript (NM_001033680.2). pFUGW-GFP was used to identify transfected neurons in spine density analysis.

Kay Y, & Herring B.E. (2021). An optogenetic method for investigating presynaptic molecular regulation. Scientific Reports, 11, 11329.

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COMMD1 Knockdown and Overexpression

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Cells were transfected with siRNA targeting COMMD1; #1 (CACCUGUUGCCAUUAUAGA[dT][dT] MISSION^® siRNA, Sigma-Aldrich) or COMMD1 siRNA #2 (CUGUUGCCAUUAUAGAGCU[dT][dT] MISSION^® siRNA, Sigma-Aldrich, St. Louis, MO, USA) to downregulate COMMD1 levels. A control siRNA (MISSION^® siRNA Universal Negative Control #1, Sigma-Aldrich, SIC001) was used in parallel. siRNA was transfected using RNAiMax (Life Technologies, 13778500) according to the manufacturer’s instructions and cells were analysed 48 to 72 h after transfection. Cells were transfected using FuGENE^® HD (Promega, E2312, Madison, WI, USA) according to the manufacturer’s instructions, with a COMMD1-FLAG siRNA-resistant plasmid (resistant to siRNA #2), which was cloned into the expression vector pcDNA3.1+/C-(K)-DYK (GenScript, Piscataway, NJ, USA). Cells were analyzed 24 h after transfection.

Suraweera A., Duijf P.H., Jekimovs C., Schrobback K., Liu C., Adams M.N., O’Byrne K.J, & Richard D.J. (2021). COMMD1, from the Repair of DNA Double Strand Breaks, to a Novel Anti-Cancer Therapeutic Target. Cancers, 13(4), 830.

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Yeast expression of human transporters

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Yeast expression constructs of human AQP9 and codon-optimized PfFNT in pDR196 (Addgene #36029) have been described before [7 (link),10 (link)]. DNA encoding the open reading frame of human MCT1 (GenBank NM_001166496) in pcDNA3.1+/C-(K)DYK was obtained commercially (GenScript, Leiden, Netherlands), and cloned into pDR196 using PflMI and BspEI restriction sites. Respective restriction sites were generated by PCR. All constructs were designed to carry an N-terminal hemagglutin epitope (HA-tag) and a C-terminal His₁₀-tag, and checked by sequencing. The Saccharomyces cerevisiae yeast strain W303-1A jen1Δ ady2Δ (MATa, can1-100, ade2loc, his3-11-15, leu2-3,-112, trp1-1-1, ura3-1, jen1::kanMX4, ady2::hphMX4) lacking endogenous monocarboxylate transporters was kindly provided by M. Casal [19 (link)]. Transformation was done using the lithium acetate/single stranded carrier DNA/polyethylene glycol method [20 (link)]. Transformed cells were grown at 29 °C in selective medium (synthetic drop-out, SD) complemented with adenine, histidine, leucine and tryptophan, and 2% (w/v) glucose, without uracil. For the phenotypical assay of yeast cell growth, 2% l-lactate was used instead of glucose. The agar media were buffered to pH 5.8 and pH 6.8, respectively, with 50 mM MES.

Bader A, & Beitz E. (2020). Transmembrane Facilitation of Lactate/H+ Instead of Lactic Acid Is Not a Question of Semantics but of Cell Viability. Membranes, 10(9), 236.

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Generation of HIP1R Point Mutants

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Human HIP1R (Huntingtin Interacting Protein 1 Related) (Accession No. NM_003959.3; 3,204 bp ORF sequence) in pcDNA3.1+/C-(K)-DYK was purchased from GenScript (NJ, USA). For constructing the point mutants of the putative Pin1 binding serine929-proline site, Ser929 was substituted by alanine (S929A) using inverse PCR method with primer sets, as described before (Suizu et al., 2016 (link)). In brief, whole plasmid DNA was amplified by the polymerase chain reaction of 16 cycles with primer sets described below in Universe Hot Start High-Fidelity 2x PCR Master mix (Biotool). After the reaction, template plasmid DNAs (wild type) were digested by DpnI enzyme (NEB). The amplified mutated linear DNA fragments were self-ligated and circulized in the presence of T4 Polynucleotide kinase (NEB) and T4 DNA ligase (NEB). The mutation site of plasmid DNA was confirmed by sanger DNA sequencing analysis in DF/HCC DNA resource core facility. Protein expression was analyzed by immunoblotting with anti-FLAG (M2) antibody (Sigma). Primer sets for S929A, sense primer; 5’-CCCCCCACCTGAGCCGC-3’, anti-sense primer5’-; CGTGCTTGTTGGCCTTCACCTTGG-3’, Primer sets for S1017A, sense primer; 5’-cCCCTGGAGAGGAGGTGGCC −3’, anti-sense primer; 5’-cGCCTGATGCCCCAGCCAG-3’

Koikawa K., Kibe S., Suizu F., Sekino N., Kim N., Manz T.D., Pinch B.J., Akshinthala D., Verma A., Gaglia G., Nezu Y., Ke S., Qiu C., Ohuchida K., Oda Y., Lee T.H., Wegiel B., Clohessy J.G., Sanatagata S., Wulf G.M., Hidalgo M., Muthuswamy S., Nakamura M., Gray N.S., Zhou X.Z, & Lu K.P. (2021). Targeting Pin1 Renders Pancreatic Cancer Eradicable by Synergizing with Immunochemotherapy. Cell, 184(18), 4753-4771.e27.

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Generating Cdx-Overexpressing Neural Crest Cells

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Plasmids (pcDNA3.1+/C-(K)-DYK) encoding full-length mouse Cdx1, Cdx2, and Cdx4 were obtained from Genscript. O9-1 mouse cranial neural crest cells (CNCC, SCC049, Millipore Sigma) were transfected with Cdx constructs using EndoFectin Max transfection reagent (GeneCopoeia, Rockville, MD, USA). The transfected cells were split after 48 h and selected for stable plasmid integration using G418 antibiotic (Gibco, ThermoFisher Scientific, Waltham, MA, USA) for 8–10 days. Resistant cell colonies were subcultured and overexpression ascertained by q-PCR analysis before use in the experiments.

Gopinathan G., Zhang X., Luan X, & Diekwisch T.G. (2023). Changes in Hox Gene Chromatin Organization during Odontogenic Lineage Specification. Genes, 14(1), 198.

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Transfection of NUDT5 ORF Plasmid

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NUDT5 ORF cDNA plasmid (OHu06714) and vector control (pcDNA3.1+/C-(K)-DYK) were purchased from GenScript (Piscataway, NJ). X-treme-Gene9 transfection reagent (XTG9-RO; Roche) was used for the transfection according to the manufacturer's instructions.

Qian J., Ma Y., Tahaney W.M., Moyer C.L., Lanier A., Hill J., Coleman D., Koupaei N., Hilsenbeck S.G., Savage M.I., Page B.D., Mazumdar A, & Brown P.H. (2024). The novel phosphatase NUDT5 is a critical regulator of triple-negative breast cancer growth. Breast Cancer Research : BCR, 26, 23.

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Ectopic Expression and Knockdown of AKAP9

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Flag-AKAP9 used for ectopic expression of AKAP9 (OHu26045) and pcDNA3.1+/C-(K)-DYK used as empty vector (EV) were purchased from GenScript (Nanjing, China). Lentiviral shAKAP9 vectors (TRCN0000232465 and TRCN0000232463) were purchased from Sigma-Aldrich. Lentiviral shGFP vector was obtained from Addgene (#30323, Watertown, MA).

Yan Q., Wu Y., Li D, & Li Y. (2022). A-Kinase Anchoring Protein 9 Promotes Gastric Cancer Progression as a Downstream Effector of Cadherin 1. Journal of Oncology, 2022, 2830634.

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Generating DDHD2 Mutant Constructs

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A wild‐type (WT) human DDHD2 expression plasmid with FLAG‐tagged in the C‐terminal region (backbone: pcDNA3.1⁺/c‐(K)‐DYK; clone ID: OHu23306) was purchased from GenScript (Piscataway, NJ, USA). The mutations, including c.335G>A (p.R112Q), c.1818C>A (p.Y606*), and c.1978G>C (p.D660H), were incorporated into the WT construct, respectively, by PCR‐based site‐directed mutagenesis method using Pfu Turbo DNA polymerase (Agilent, Santa Clara, CA, USA). Given that the p.Y606* mutation may lead to premature truncation of the protein product, the C‐terminal FLAG‐tag (DYKDDDDK) of all DDHD2 constructs (WT and mutants) was moved to the N‐terminal using Q5 Site‐Directed Mutagenesis Kit according to the manufacturer's protocol (New England Biolabs, Ipswich, MA, USA).
Human embryonic kidney 293T (HEK293T) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) and supplemented with 10% fetal bovine serum at 37°C under 5% CO₂. Transient transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Chou Y., Hsu S., Tsai Y., Lu Y., Yu K., Wu H., Liao Y, & Lee Y. (2023). Biallelic DDHD2 mutations in patients with adult‐onset complex hereditary spastic paraplegia. Annals of Clinical and Translational Neurology, 10(9), 1603-1612.

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C3 Expression and Cardiomyocyte Viability

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The AC16 human cardiomyocyte cell line (Millipore, MA, USA) was grown in DMEM:F12 with 10% heat-inactivated FBS, 1% penicillin, and 1% L-Glutamine. The 293 T cell line was grown in DMEM with 10% heat-inactivated FBS and 1% penicillin. Both cell lines were maintained at 37 °C in 5% CO₂. Rabbit anti-human C3c (F0201) polyclonal Ab was obtained from Agilent Dako (Santa Clara, CA, USA). Goat anti-human C3 (A213) polyclonal Ab was obtained from Complement Technologies (Tyler, TX, USA). Rabbit anti-cytochrome c polyclonal Ab was obtained from Cell Signaling (Danvers, MA, USA). Rabbit anti-caspase 3 polyclonal Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The full length human complement C3 expression ORF clone, whose C3 gene cDNA ORF clone sequences were retrieved from the NCBI Reference Sequence Database with the vector pcDNA 3.1+/C-(K)DYK, was obtained from GenScript (Piscataway, NJ, USA) (Supplementary Figure S4).

Fang Z., Lee H., Liu J., Wong K.A., Brown L.M., Li X., Xiaoli A.M., Yang F, & Zhang M. (2023). Complement C3 Reduces Apoptosis via Interaction with the Intrinsic Apoptotic Pathway. Cells, 12(18), 2282.

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