Pcdna3

Manufactured by GenScript

Sourced in China, United States

pCDNA3.1 is a mammalian expression vector designed for the high-level expression of recombinant proteins in a variety of cell lines. It contains a cytomegalovirus (CMV) promoter, the neomycin resistance gene for selection, and a polyadenylation signal.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (16 mentions) Pcdna3, by Thermo Fisher Scientific (11 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Mir nc, by GenePharma (3 mentions) In fusion, by Takara Bio (2 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (2 mentions) Human acth 1 24, by Phoenix Pharmaceuticals (2 mentions) X tremegene hp dna transfection reagent, by Merck Group (2 mentions) Neb phusion high fidelity master mix, by New England Biolabs (2 mentions) Nebuilder hifi dna assembly master mix, by New England Biolabs (2 mentions) Si nc, by GenePharma (2 mentions) Mimic nc, by RiboBio (2 mentions) Q5 high fidelity dna polymerase, by GenScript (2 mentions) Purelink genomic dna mini prep kit, by Thermo Fisher Scientific (2 mentions) Xl1 blue, by Agilent Technologies (2 mentions) Midiprep kit, by Qiagen (2 mentions) Mir con, by RiboBio (2 mentions) Pci neo, by Promega (2 mentions) Lipofectamine, by Promega (2 mentions)

Close spelling variants of this product

PcDNA3.1 vector (66 citations) PcDNA3.1+/C-(K)-DYK vector (18 citations) PcDNA3.1/Zeo (+)) (4 citations) PcDNA3.1-P2A-eGFP vector (4 citations) PcDNA3.1+/C-(K)DYK-ACE2 (3 citations) PcDNA3.1/Hygro (3 citations) PcDNA3.1+N-terminal FLAG (DYK) tag expression vector (2 citations) PcDNA3.1-PGAM1-FLAG (2 citations) PcDNA3.1 expression plasmids (2 citations) PcDNA3.1‐P2A‐eGFP (2 citations)

90 protocols using pcdna3

Aspartoacylase Expression and Variants

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Full-length wild-type human ASPA cDNA with an N-terminal RGS6xHis-tag was expressed from pcDNA3.1 (Genscript). The C152W variant was generated by Genscript. The empty vector control was kindly provided by Dr. Mads Gyrd-Hansen. Full-length codon-optimized yeast HSP104 fused C-terminally to an HA-tag was expressed from pcDNA3.1 (Genscript). Human codon-optimized HSPH1 fused C-terminally to a Myc-tag was expressed from pcDNA3.1 (Genscript). cDNA encoding wild-type human ubiquitin containing an N-terminal HA-tag was expressed from pRK5-HA (Addgene). Full-length EGFP fused N-terminally to a nuclear localization signal (NLS) and C-terminally to a Myc-tag was expressed from pCMV (Clontech).
In yeast, wild-type codon-optimized ASPA cDNA fused to Ura3-HA-GFP at the N-terminal of ASPA was expressed from pTR1412 (Genscript). The pTR1412 vector was kindly provided by Dr. Tommer Ravid. The ASPA fusion constructs were used for all yeast experiments. The C152W variant was generated by Genscript. The ASPA degron sequences fused N-terminally to Ura3-HA-GFP were expressed from pTR1412, and the sequences were RNNFLIQMFHYIKTSLAPLPCYVYLIEHP (Degron wt) and RNNFLIQMFHYIKTSLALPWYVYLIEHP (Degron C152W) (Genscript).

Gersing S.K., Wang Y., Grønbæk-Thygesen M., Kampmeyer C., Clausen L., Willemoës M., Andréasson C., Stein A., Lindorff-Larsen K, & Hartmann-Petersen R. (2021). Mapping the degradation pathway of a disease-linked aspartoacylase variant. PLoS Genetics, 17(4), e1009539.

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Codon-Optimized DPR Constructs

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Codon optimized cDNA constructs encoding the C9orf72 DPRs (polyGA x40 , polyGR x40 , polyGP x40 , polyPR x40, polyPA x40 ), tagged with the FLAG epitope at the amino terminus, were subcloned into pcDNA3 (Genscript) using BamH1 and EcoRV. Expression of each DPR was driven by the presence of an initiation codon, ATG. A second set of constructs were designed containing either G4C2 x0 , G4C2 x3 and G4C2 x40 repeats, tagged with mCherry, FLAG, HA and MYC epitopes at the amino terminus, and were also subcloned using BamH1 and EcoRV into pcDNA3 (Genscript). These constructs, G4C2 RANx0, G4C2 RANx3, or G4C2 RANx40, were created without an ATG codon, so that they can perform RAN translation in any forward reading frame. The XBP-1-Venus construct was a kind gift from Dr Masayuki Miura [56] (link). The constructs encoding mutant vesicular stomatitis viral glycoprotein (VSVG ts045 ), fused to either mCherry in pEGFPdKA206K-N1-mCherry vector or VSVG ts045 fused to EGFP in pEGFP-C1, were a kind gift from Dr Jennifer Lippincott-Schwartz (National Institutes of Health, Bethesda, USA). The EGFP-Double FYVE-containing protein 1 (DFCP1) in pEGFP-C2 (Invitrogen) was a kind gift from Dr Nicholas Ktistakis, Babraham Institute, Cambridge, England, UK [52] (link).

Sultana J., Ragagnin A.M.G., Parakh S., Saravanabavan S., Soo K.Y., Vidal M., Jagaraj C.J., Ding K., Wu S., Shadfar S., Don E.K., Deva A., Nicholson G., Rowe D.B., Blair I., Yang S, & Atkin J.D. (2024). C9orf72-Associated Dipeptide Repeat Expansions Perturb ER-Golgi Vesicular Trafficking, Inducing Golgi Fragmentation and ER Stress, in ALS/FTD. Molecular neurobiology.

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Cytotoxicity Evaluation of Sorafenib and Regorafenib in Liver Cancer Cells

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HepG2, Hep3B and HuH7 cells were seeded into six-well plates at a density of 3×10⁵ cells/well. When reaching 30% confluence, cells were transfected with overexpression plasmids or siRNA using the jetPRIME reagent (Polyplus-transfection SA) according to the manufacturer's instructions. siRNAs against human DYRK1A and HIF-1α were synthesized by Shanghai GenePharma Co., Ltd. (20 (link)). The sense sequences of human siRNAs were as follows: siDYRK1A-1, 5′-AUG GAG CUA UGG ACG UUA ATT-3′; siDYRK1A-2, 5′-AAA CUC GAA UUC AAC CUU ATT-3′; siHIF-1α-1, 5′-CUG AUG ACC AGC AAC UUG ATT-3′; siHIF-1α-2, 5′-AUC CAG AGU CAC UGG AAC UTT-3′; and negative control siRNA (siNC), 5′-UUC UCC GAA CGU GUC ACG UTT-3′. The plasmid pcDNA3.1 containing the insert DYRK1A and empty vector pcDNA3.1 were purchased from GenScript. After 24 h siRNA transfection, the cells were treated with 2.5-20 µM sorafenib and/or 0.25-8 µM regorafenib (ranging from 0.25-8 µM) at 37°C for 72 h. SRB was used to detect the cytotoxicity of cancer cells.

Zhang C., Wu L.W., Li Z.D., Zhang M.M., Wu J., Du F.H., Zeng L.H, & Li Y.L. (2022). DYRK1A suppression attenuates HIF-1α accumulation and enhances the anti-liver cancer effects of regorafenib and sorafenib under hypoxic conditions. International Journal of Oncology, 60(4), 45.

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Engineered SARS-CoV-2 Spike Protein and CR3022 Fab

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A construct coding for the SARS-CoV-2 spike glycoprotein ectodomain (YP_009724390.1, residues 1-1208) was codon-optimised for human expression, synthesised, and cloned into pcDNA.3.1(+) vector by GenScript. The construct was made with a “FUR 2P” set of stabilising mutations:^{15 (link)} R682S and R685S, which removed the polybasic cleavage site between S1 and S2; and K986P and V987P, which inhibited the propensity of the spike to assume the post-fusion conformation. The spike protein had a signal peptide derived from µ-phosphatase attached at the N terminus and a TEV-cleavage site, a foldon trimerisation motif, and a hexahistidine tag, all separated by short linkers, added at the C terminus.
The sequences coding for the variable regions of heavy (DQ168569.1) and light chains (DQ168570.1) of the CR3022 were added to those of the human constant regions to yield constructs coding for heavy and light chains of a Fab. The heavy chain construct had a human Ig heavy chain signal peptide at its N terminus for secretion and hexahistidine tag preceded by a TEV-cleavage site at its C terminus for purification; the light chain construct had a human Ig kappa chain signal peptide added at its N terminus. The constructs were synthesised with codons optimised for human expression by GenScript and cloned independently into pcDNA.3.1(+) vectors.

Wrobel A.G., Benton D.J., Hussain S., Harvey R., Martin S.R., Roustan C., Rosenthal P.B., Skehel J.J, & Gamblin S.J. (2020). Antibody-mediated disruption of the SARS-CoV-2 spike glycoprotein. Nature Communications, 11, 5337.

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Diverse Cas13 Variants for CRISPR Applications

Cited 2 times

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Cas13bt, EsCas13d, RspCas13d and PspCas13b were obtained from Addgene (#176316, #108303, #108305, and #103862, respectively)^{7 (link),16 (link),23 (link)}. EsCas13d, RspCas13d, and PspCas13b sequences were amplified by PCR and cloned into a pcDNA3.1 backbone. Cas13X (#171379) and REPAIRx were gifts from Professor Hui Yang and Xingxu Huang, respectively^{15 (link),38 (link)}. Mini-RfxCas13d, mini-EsCas13d, mini-RspCas13d, mini-PspCas13b, mini-PbuCas13b, PbuCas13b and mini-Vx were synthesized and cloned into pcDNA3.1(+) (GenScript Biotech, China). The Δ1, Δ2, and Δ3 RfxCas13d strains were constructed by homologous recombination. Δ4, Δ5, Δ6, Δ7, and Δ8 were synthesized and cloned into pcDNA3.1(+) by GenScript Biotech (China). The sequences of the proteins are listed in Table S1. All of the primers used for plasmid construction are listed in Table S2.

Zhao F., Zhang T., Sun X., Zhang X., Chen L., Wang H., Li J., Fan P., Lai L., Sui T, & Li Z. (2023). A strategy for Cas13 miniaturization based on the structure and AlphaFold. Nature Communications, 14, 5545.

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Manipulating miR-27b and HOXB8 in OS Cells

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To manipulate miR-27b expression in OS cell lines, synthetic miR-27b mimic
(5′-AGAGCUUAGCUGAUUGGUGAAC-3′) and the corresponding negative control (NC-miR,
5′-GATAGGCGTACTGAGTACTTGA-3′) purchased from GenePharm (Shanghai, China) were utilized. To
control the expression of HOXB8, the pcDNA3.1 containing open reading frame of HOXB8
(pHOXB8) and the empty vector pcDNA3.1 purchased at GenScript (Nanjing, Jiangsu, China)
were used. Cells transfection was conducted using Lipofectamine 2000 (Invitrogen) when
cells cultured to approximately 70% to 80% of confluence.

Wu Y, & Peng J. (2019). miR-27b Targets HOXB8 to Inhibit Malignant Behaviors of Osteosarcoma. Technology in Cancer Research & Treatment, 18, 1533033819870791.

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Characterization of FLCN Interactions

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Full-length wild-type human FLCN cDNA carrying an N-terminal RGS6xHis-tag was expressed from pcDNA3.1 (Genscript). USP7 was expressed with an N-terminal myc-tag from pcDNA3.1 (Genscript). All mutations were generated by Genscript. The pcDNA3.1-FLAG-FNIP1 and the pcDNA3.1+N-eGFP-FLCNΔE510 vectors were generated by Genscript. The pIRES2-FLCN plasmids were kindly provided by Dr. E. R. Maher (Birmingham, UK). The pEGFP-N1-FLCN, pRK5-HA-FNIP1 and pRK5-HA-FNIP2 constructs were kindly provided by Dr. D. M. Sabatini (MIT, USA).

Clausen L., Stein A., Grønbæk-Thygesen M., Nygaard L., Søltoft C.L., Nielsen S.V., Lisby M., Ravid T., Lindorff-Larsen K, & Hartmann-Petersen R. (2020). Folliculin variants linked to Birt-Hogg-Dubé syndrome are targeted for proteasomal degradation. PLoS Genetics, 16(11), e1009187.

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Functional Characterization of SLC9A3-AS1 and miR-486-5p

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Small interfering RNAs (siRNAs) synthesized against SLC9A3-AS1 (si-SLC9A3-AS1) and a corresponding negative control (NC) siRNA (si-NC) were obtained from Shanghai GenePharma Co., Ltd. The si-SLC9A3-AS1#1 sequence was 5′-CACATGTTTTTTATAATAAAACA-3′; the si-SLC9A3-AS1#2 sequence was 5′-ATGTTTTTTATAATAAAACATAG-3′; and the si-NC sequence was 5′-CACGATAAGACAATGTATTT-3′. The miR-486-5p mimic, miR-486-5p inhibitor, and the matching controls (NC mimic and NC inhibitor, respectively) were prepared by Guangzhou RiboBio Co., Ltd. The miR-486-5p mimic sequence was 5′-GAGCCCCGUCGAGUCAUGUCCU-3′ and the NC mimic sequence was 5′-UUGUACUACACAAAAGUACUG-3′. The miR-486-5p inhibitor sequence was 5′-CUCGGGGCAGCUCAGUACAGGA-3′ and the NC inhibitor sequence was 5′-ACUACUGAGUGACAGUAGA-3′. The pcDNA3.1-E2F6 plasmid was constructed by cloning the E2F6 cDNA into pcDNA3.1 (GenScript Biotech Corporation). The transfection of siRNA (100 pmol), miRNA oligonucleotides (100 pmol) or plasmids (4 µg) was performed at room temperature with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 24 h post-transfection, Cell Counting Kit-8 (CCK-8), colony-forming and cell migration and invasion assays were performed. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), flow cytometry and western blotting were carried out after 48 h of culture.

Li J., Li D., Zhang X., Li C, & Zhu F. (2021). Long noncoding RNA SLC9A3-AS1 increases E2F6 expression by sponging microRNA-486-5p and thus facilitates the oncogenesis of nasopharyngeal carcinoma. Oncology Reports, 46(2), 165.

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Optimized SARS-CoV-2 Spike Protein Expression

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The spike coding sequences for SARS-CoV-2 lineage A (nCoV-WA1–2020) and variant B.1.1.7 (hCoV320 19/England/204820464/2020, EPI_ISL_683466) were truncated by deleting 19 aa at the C-terminus. The S proteins with the 19 aa deletion of coronaviruses were previously reported to show increased efficiency regarding incorporation into virions of VSV^{46 (link),47 (link)}. These sequences were codon optimized for human cells, then appended with a 5′ kozak expression sequence (GCCACC) and 3′ tetra-glycine linker followed by nucleotides encoding a FLAG-tag sequence (DYKDDDDK). These spike sequences were synthesized and cloned into pcDNA3.1⁺(GenScript). Human and hamster ACE2 (Q9BYF1.2 and GQ262794.1, respectively), were synthesized and cloned into pcDNA3.1⁺ (GenScript). All DNA constructs were verified by Sanger sequencing (ACGT).

Port J.R., Yinda C.K., Avanzato V.A., Schulz J.E., Holbrook M.G., van Doremalen N., Shaia C., Fischer R.J, & Munster V.J. (2021). Increased aerosol transmission for B.1.1.7 (alpha variant) over lineage A variant of SARS-CoV-2. bioRxiv.

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Regulating RUNX2 Expression with miR-205

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miR-205 mimic (5′-UCCUUCAUUCCACCGGAGUCUG-3′) and control (miR-con, 5′-GGUCCGUCCGUAAUUAUCCUCC-3′) oligonucleotides were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). RUNX2 small interfering RNA (siRNA, 5′-AAGGACAGAGTCAGATTACAG-3′) and control (si-con, 5′-ATAAGGTATCGAGACCAGAGA-3′) oligonucleotides were also purchased from Guangzhou RiboBio Co., Ltd. The RUNX2 open reading frame cloned into pcDNA3.1 vector and the empty vector pcDNA3.1 were purchased from GenScript (Nanjing, China). Transfection was conducted with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol (100 nM miRNAs and siRNAs, 5 µg RUNX2 construct and empty vector). Following transfection for 48 h, the cells were used for subsequent experiments.

Zhuang L., Guo J., Yao Y, & Li Z. (2018). miR-205 targets runt-related transcription factor 2 to inhibit human pancreatic cancer progression. Oncology Letters, 17(1), 843-848.

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