Pcdna3

Manufactured by Addgene

Sourced in United States, China

The pcDNA3.1 is a mammalian expression vector used for the expression of recombinant proteins in various cell lines. It contains a human cytomegalovirus (CMV) immediate-early promoter for high-level expression and a neomycin resistance gene for selection in mammalian cells.

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Lab products found in correlation

Spelling variants of this product

FLAG-HA-pcDNA3.1 (25 citations) PcDNA3 Myr-HA Akt1 (7 citations) PcDNA3/Myc-DNMT3B1 (4 citations) PcDNA3.3-hCas9 (2 citations) PCDNA3.1 FLAG NRF2 (2 citations) 3xHA-mTurbo-NLS_pCDNA3 (2 citations) PcDNA3-enhanced green fluorescent protein (2 citations) PcDNA3-HA-ubiquitin plasmid (2 citations) HA-GSK3 β S9A pcDNA3 (2 citations) SnailHA_pcDNA3 (2 citations)

119 protocols using pcdna3

Overexpression and Silencing of Molecular Targets

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Empty vector pcDNA3.1 (Catalog no. V790-20) was purchased from Thermo Fisher Scientific Company. cDNAs that were used for construction of overexpression plasmid of lnc-HZ09 (pcDNA3.1-HZ09), METTL3 mRNA (pcDNA3.1-METTL3), MSX1 mRNA (pcDNA3.1-MSX1), SP1 mRNA (pcDNA3.1-SP1), or HuR mRNA (pcDNA3.1-HuR) were synthesized and constructed into pcDNA3.1 vector by Addgene (Table S1). The corresponding RNA sequences were obtained from National Center for Biotechnology Information (NCBI) database (Gene Bank, Homo sapiens, GRCh38.p14; sequences in Table S1). Empty vector pcDNA3.1 was used as a negative control. Si-HZ09, si-METTL3, si-MSX1, si-SP1, si-HuR, and si-NC (negative control) were customized by Thermo Fisher (sequences in Table S2). Swan 71 and HTR-8/SVneo cells (

1 times 10 begin superscript 6 end superscript

cells/well) were seeded in 6-well plates and cultured to 80% confluence. Trophoblast cells were transfected with

2.0 micrograms per well

plasmids or

50 nanomolar

siRNAs in Lipofectamine 3,000 (Invitrogen) medium for 24 h according to the manufacturer’s protocols. For all assays, cell quantification was performed using TC20 Automated Cell Counter (Bio-Rad Laboratories).

Dai M., Huang W., Huang X., Ma C., Wang R., Tian P., Chen W., Zhang Y., Mi C, & Zhang H. (2023). BPDE, the Migration and Invasion of Human Trophoblast Cells, and Occurrence of Miscarriage in Humans: Roles of a Novel lncRNA-HZ09. Environmental Health Perspectives, 131(1), 017009.

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Cre-Dependent PSD-95-mCherry Expression

Cited 2 times

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The Cre-dependent eYFP and Teal-gephyrin plasmids (pFUdioeYFPW, pFUdioTealGephyrinW) have been described previously in (Chen et al., 2012 (link)), and the Cre plasmid in (Subramanian et al., 2013 (link)). To generate a Cre-dependent PSD-95-mCherry within a dio cassette, PSD-95 lacking a stop codon was first amplified with an added 5’Nhe1 site from pFuPSD-95-TealW (gift from Jerry Chen), then cloned into pcDNA3 (Invitrogen) between the KpnI and EcoRI sites, to create pcDNA3-PSD-95. mCherry was amplified from pcDNA3.3-mCherry (Addgene) with an added 3’AgeI site, then cloned into pcDNA3-PSD-95 between the EcoRI and XhoI sites to make pcDNA3-PSD-95-mCherry. PSD-95-mCherry was then removed by NheI and AgeI digestion and subcloned into the Cre dependent plasmid pFudio-AscI-AgeI-NheIW, generated by replacing eYFP in pFudio-eYFPW with a linker sequence containing the AgeI restriction site. The vector backbone used for all our expression constructs is the lentivirus transfer vector pFUGW. This is not a strong expression vector and therefore likely expresses at lower levels than more conventional expression constructs. This is an advantage when expressing synaptic markers, and explains the lack of synaptic artifacts frequently seen in other systems, in particular with PSD-95 expression, such as increases in synapse number or stability.

Villa K.L., Berry K.P., Subramanian J., Cha J.W., Oh W.C., Kwon H.B., Kubota Y., So P.T, & Nedivi E. (2016). Inhibitory synapses are repeatedly assembled and removed at persistent sites in vivo. Neuron, 89(4), 756-769.

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Expressing Viral Gag Proteins Using Plasmids

Cited 1 time

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To express viral Gag proteins, a MuLV Gag/Pol expression vector AKAQ188 [14 (link)] and an HIV-1-based packaging vector pCMV-dR8.74 were used. The following plasmids were obtained from Addgene (Watertown, MA, USA): pArf6-CFP (#11382) [15 (link)], pArf6(Q67L)-CFP (#11387) [15 (link)], pcDNA3 HA-Arf6 (#10834) [16 (link)], pcDNA3 HA-Arf6 ActQ67L (#10835) [16 (link)], 2PH-PLCdelta-GFP (#35142) and CD63-pEGFP C2 (#62964). The plasmids pcDNA3 (Invitrogen, Carlsbad, CA, USA) and pEGFP-N1 (TaKaRa Bio, San Jose, CA, USA) were used as transfection controls and were added to equalize the total amounts of DNA transfected.

Kang H., Kang T., Jackson L., Murphy A, & Nitta T. (2024). Evidence for Involvement of ADP-Ribosylation Factor 6 in Intracellular Trafficking and Release of Murine Leukemia Virus Gag. Cells, 13(3), 270.

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Smurf1 Mutant Expression and Purification

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A cDNA construct containing the full-length open reading frame of Smurf1 wild type was subcloned into Flag-tagged mammalian expression vector pcDNA3.1 (Addgene). Site mutations including R289A, G248A, Y297A and G248A/Y297A in Flag-Smurf1 were made by the QuikChange site-directed mutagenesis kit (Agilent Technologies) and subcloned into Flag-tagged mammalian expression vector pcDNA3.1 (Addgene). Both Smurf1 wild type and mutants were verified by DNA sequencing analysis. The expression vectors were transfected into mouse osteoblasts using X-tremeGENE^™ HP DNA Transfection Reagent (Roche Life Science), according to the manufacturer’s instructions. Flag-tagged Smurf1 wild type and mutants were purified by a FLAG® M purification kit (Sigma), according to the manufacturer’s instruction.

Liang C., Peng S., Li J., Lu J., Guan D., Jiang F., Lu C., Li F., He X., Zhu H., Au D.W., Yang D., Zhang B.T., Lu A, & Zhang G. (2018). Inhibition of osteoblastic Smurf1 promotes bone formation in mouse models of distinctive age-related osteoporosis. Nature Communications, 9, 3428.

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CCAT1 Cloning and Mutagenesis Protocol

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The complementary DNA encoding CCAT1 was PCR-amplified by the Pfu Ultra II Fusion HS DNA Polymerase (Stratagene, Agilent Technologies, Palo Alto, CA, USA) and subcloned into the pcDNA3.1 vector (Invitrogen). The primers used were 5′-CTAGCTAGCACAACATCGACTTTGAAGTT-3′ (forward) and 5′-CCCAAGCTTAAGACTTAATATACTTATATTTA-3′ (reverse). The pcDNA3.1-CCAT1 with point mutations in let-7 binding sites was synthesized by GenScript (Nanjing, China) and named pcDNA3.1-CCAT1-Mut. pSL-MS2-12X (Addgene) was double digested with BamH I and Xba I, and the MS2-12X fragment was subcloned into pcDNA3.1, pcDNA3.1-CCAT1 or pcDNA3.1-CCAT1-Mut, named pcDNA3.1-MS2, pcDNA3.1-MS2-CCAT1 or pcDNA3.1-MS2-CCAT1-Mut respectively. The let-7 binding region of either lncRNA-CCAT1 or lncRNA-CCAT1-Mut was amplified using PCR and subcloned into the pmirGLO vector (Promega, Madison, WI, USA) for Luciferase reporter assay. The primers used were 5′-CGAGCTCTCAACCCTGACGCTCTTTCTG-3′ (forward) and 5′-GCTCTAGATCACTCACCCACTGCTCACC-3′ (reverse).

Deng L., Yang S.B., Xu F.F, & Zhang J.H. (2015). Long noncoding RNA CCAT1 promotes hepatocellular carcinoma progression by functioning as let-7 sponge. Journal of Experimental & Clinical Cancer Research : CR, 34(1), 18.

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Investigating CDK5 Regulation of NF-κB

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Neurobasal medium, B27 supplement, high-glucose Dulbecco’s Modified Eagle Medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). pcDNA3.0, pcDNA-CDK5, and GFP-p25 plasmids were obtained from Addgene (Cambridge, MA, USA). An NF-κB luciferase reporter plasmid was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Lipofectamine 2000 transfection reagent and Opti-MEMI medium were obtained from Invitrogen (Carlsbad, CA, USA). Etoposide and roscovitine were obtained from Sigma-Aldrich (St. Louis, MO, USA). A luciferase reporter gene assay kit was purchased from Roche (Basel, Switzerland). The 96-well plate used for the luciferase reporter gene test was purchased from Greiner (Lud-wigsburg, Germany). Other cell culture plates were purchased from Corning (Corning, NY, USA). The primary antibody against caspase 1 was obtained from Abcam (Cambridge, MA, USA). Primary antibodies, including anti-CDK5, anti-phosphorylated (p)-CDK5, anti-IL-1β, and anti-β-actin antibodies, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-rabbit and goat-anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Jackson Immuno Research (West Grove, PA, USA).

Sun H., Cai J., Shen S, & Ren X. (2019). Hypothermia treatment ameliorated cyclin-dependent kinase 5-mediated inflammation in ischemic stroke and improved outcomes in ischemic stroke patients. Clinics, 74, e938.

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Overexpression of Ras in Breast Cancer

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For the overexpression of Ras gene in breast cancer cells, full-length Homo sapiens Ras genomic sequences based in NCBI were cloned and ligated onto pcDNA3.0 (Addgene, Watertown, MA, USA). The recombinant constructs were sequence verified through DNA sequencing by the Thermo Fishier Scientific.

Zheng W., Cao L., Ouyang L., Zhang Q., Duan B., Zhou W., Chen S., Peng W., Xie Y., Fan Q, & Gong D. (2019). Anticancer activity of 1,25-(OH)2D3 against human breast cancer cell lines by targeting Ras/MEK/ERK pathway. OncoTargets and therapy, 12, 721-732.

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Exo70, Ubiquitin, and shRNA Lentiviral Construct Generation

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Human Exo70 was Flag, mCherry, or GFP tagged by PCR and subcloned into pCMV10 (RRID: Addgene_51888) or pLV (RRID: Addgene_85140) vector. Ubiquitin (Ub) was Myc-tagged by PCR and subcloned into pcDNA3.0 (RRID: Addgene_12452) vector. shRNA were synthesized and cloned into pLKO.1 (RRID: Addgene_52920) vector. The sequences of shRNAs were listed in Table S2. Lentiviruses were generated by transfecting 293T cells with pLKO.1 vector encoding shRNA or pLV vector encoding Exo70, together with package vectors pMDL, pVSVG, and pRSV-Rev (RRID: Addgene_12253). Viruses were collected and infected cells. Stable cell lines were established by selection of 2 μg/mL puromycin.

Zhao Y., Hong X., Chen X., Hu C., Lu W., Xie B., Zhong L., Zhang W., Cao H., Chen B., Liu Q., Zhan Y., Xiao L, & Hu T. (2021). Deregulation of Exo70 Facilitates Innate and Acquired Cisplatin Resistance in Epithelial Ovarian Cancer by Promoting Cisplatin Efflux. Cancers, 13(14), 3467.

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Analyzing Cellular Signaling Pathways

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All information regarding antibodies used in this study is provided in Table S7. Rapamycin (Rapa), everolimus (RAD001), deferoxamine (DFX), DAPT and MHY1485 were purchased from Selleck Chemicals (Houston, TX, USA). Jagged1-Fc was obtained from R&D system (Minneapolis, MN, USA). Lipofectamine RNAiMax was obtained from Invitrogen (Carlsbad, CA, USA). pRL-TK, pGL3-Basic, pcDNA3.0, pcDNA3.0-HA-HIF-1α, lenti-CRISPRv2 plasmids and packaging vectors (pVSVG and psPAX2) were purchased from Addgene (Cambridge, MA, USA).

Li D., Sun A., Zhang L., Ding Z., Yi F., Yang X., Wang Z., Chen X., Liu W., Liu S., Shen H., Miao M., Zhang L., Liu P., Liu Y., Su S., Huang H., Huang C., Hu Z., Zhang H., Zha X, & Liu Y. (2022). Elevated ITGA5 facilitates hyperactivated mTORC1-mediated progression of laryngeal squamous cell carcinoma via upregulation of EFNB2. Theranostics, 12(17), 7431-7449.

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Cloning and Mutating Human C3 Protein

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Full-length cDNA encoding human C3 was cloned into pcDNA3 (Invitrogen). AUG to AUU and C3 STOP codon mutations in human C3 sequence were introduced using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies), using primer pairs found in supplementary Table 1. Double and triple mutant C3-pcDNA plasmids were prepared from single (ΔAUG2 and ΔAUG3) or double (ΔAUG2 + 3) mutant constructs, respectively, using primer pairs which mutate the first AUG codon. All variants were confirmed by Sanger DNA sequencing (Eurofins Genomics). pET21a-BirA was purchased from Addgene (#20,857) and the BirA insert sub-cloned into pcDNA3. BAP-tagged C3 was generated by cloning of a synthesized C3-BAP fragment into WT C3-pcDNA3 using HindIII and BlpI restriction sites.

Kremlitzka M., Colineau L., Nowacka A.A., Mohlin F.C., Wozniak K., Blom A.M, & King B.C. (2022). Alternative translation and retrotranslocation of cytosolic C3 that detects cytoinvasive bacteria. Cellular and Molecular Life Sciences, 79(6), 291.

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