Egfp c2

Manufactured by Takara Bio

Sourced in United States

The EGFP-C2 is a plasmid vector that expresses the enhanced green fluorescent protein (EGFP) under the control of a CMV promoter. It can be used for the expression and detection of EGFP-tagged fusion proteins in mammalian cells.

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Lab products found in correlation

Pcdh cmv mcs ef1 puro lentiviral vector, by System Biosciences (3 mentions) Pgl2 vector, by Promega (1 mentions) Ex taq dna polymerase, by Takara Bio (1 mentions) Pgem t easy vector, by Promega (1 mentions) Neon transfection system, by Thermo Fisher Scientific (1 mentions) Lipo2000, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Pegfp c2, by Takara Bio (1 mentions) Egfp c1 vector, by Takara Bio (1 mentions) Gcamp6f, by Addgene (1 mentions) Egfp c1, by Takara Bio (1 mentions) Egfp c3, by Takara Bio (1 mentions)

7 protocols using egfp c2

Piccolo and Daam1 Cloning Protocols

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The Piccolo_1980-2553 clone [44 (link)] has flanking EcoRI sites that were used to clone the sequence into the EcoRI sites of EGFP C2 (Clontech) and the mRFP ActA vector (kindly provide by W.J. Nelson Stanford University). A NotI—Piccolo_1980-2553-Stop-XhoI fragment was generated by PCR and cloned into the CD4 construct (kindly provide by Chen Gu, Ohio State University). The EGFP coding sequence with flanking NotI sites (NotI-EGFP-NotI) was then cloned into the NotI site to obtain CD4-NotI-EGFP-NotI- Piccolo_1980-2553-Stop-XhoI. A control plasmid CD4-NotI-EGFP-NotI-Stop-XhoI was constructed similarly.
Mouse Daam1 cDNA (Accession no. AY426535) was kindly provided by Dr. Terry Yamaguchi, NCI. All Daam1 fragments were PCR amplified from original mouse Daam1 cDNA and cloned directionally into XhoI-SmaI sites of EGFP C1, EGFP C2, or EGFP C3 vectors (all from Clontech) as required. The sequence for the EGFP tag was then excised and replaced with oligonucleotides containing the sequence for the Myc epitope tag. Please see supplementary information (S1 Table) for primer sequences.

Wagh D., Terry-Lorenzo R., Waites C.L., Leal-Ortiz S.A., Maas C., Reimer R.J, & Garner C.C. (2015). Piccolo Directs Activity Dependent F-Actin Assembly from Presynaptic Active Zones via Daam1. PLoS ONE, 10(4), e0120093.

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Tri-reporter Vector Construction and Characterization

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The tri-reporter was constructed by linking the coding sequences of firefly luciferase, eGFP, and HSV1Δtk, which has a truncation at the HSV1tk N-terminus. As previously described^{28 (link)}, the HSV1tk (GenBank: CAA23742) coding sequence (a.a. 1–376) was inserted into the C-terminus of an eGFP expression vector (EGFP-C2, Clontech) at the EcoR1/BamH1 sites. The first 135 bp of HSV1tk in the eGFP-tk vector was removed by PCR-directed mutagenesis, resulting in the eGFP-Δtk vector. The F1B promoter, which was isolated from an F1B-GFP vector^{12 (link),26 (link)} as a 540-bp ApaL1/Age1 fragment, was used to replace the CMV promoter in the eGFP-Δtk vector, resulting in the F1B-eGFP-Δtk vector. The coding sequence of firefly luciferase (Luc) was isolated from the pGL2 vector (Promega) by PCR and inserted into the F1B-eGFP-Δtk vector at the Age1 site to generate the F1B-Luc-eGFP-Δtk vector (F1B-TMIR). Luc was similarly inserted into the eGFP-Δtk vector to generate the CMV-TMIR vector. All vectors were verified by DNA sequencing.

Liu S.W., Hsu C.H., Chen M.R., Chiu I.M, & Lin K.M. (2019). A Tri-fusion Reporter Mouse Reveals Tissue-Specific FGF1B Promoter Activity in vivo. Scientific Reports, 9, 11143.

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Construction of hEF1α-EGFP and CMV-EGFP Plasmids

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To construct hEF1α-EGFP and CMV-EGFP plasmid constructs, LCMV:ECFP(loxP)(FRT)EYFP (#31304; Addgene, MA, USA) was modified by gene cloning. EYFP sequence was eliminated using Sma1 and Kpn1. The ECFP gene was replaced with the EGFP sequence using NcoI and BsrGI from EGFP-C2 (#6083–1; Clontech Laboratories Inc., CA, USA). The following genes and elements were obtained by PCR amplification: Human EF1α promoter (from pCDH-CMV-MCS-EF1-puro; System Biosciences Inc., CA, USA), CMV promoter (from pcDNA^™-3.1⁽⁺⁾; Thermo Fisher Scientific, MA, USA). PCR amplification process was performed using Extaq^® DNA polymerase (Takara Bio Inc., CA, USA) and the following primers: hEF1α, forward (F) 5′-aggactctcactttctctctctgc-3′ and reverse (R) 5′-tgcaggctttatggaggagt-3′; CMV, (F) 5′-gggccagatatactcgttga-3′ and (R) 5′-gccagagagctctgcttat-3′. After amplification, each product was ligated to pGEM^®-T Easy Vector (Promega, WI, USA) according to the manufacturer’s instructions and verified by sequencing analysis (BIONICS Corp., Seoul, Korea). Finally, they were digested with each of the designated restriction enzymes and ligated into the plasmid vector.

Eun K., Hong N., Jeong Y.W., Park M.G., Hwang S.U., Jeong Y.I., Choi E.J., Olsson P.O., Hwang W.S., Hyun S.H, & Kim H. (2020). Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer. PLoS ONE, 15(6), e0233784.

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Overexpression of PATZ1 in Thyroid Cancer Cells

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All human thyroid carcinoma cell lines were cultured in DMEM supplemented with 10% FBS, L-glutamine, and penicillin/streptomycin (GIBCO-BRL) in a 5% CO2 atmosphere. TPC1 and FRO cells were transfected using Neon Transfection System (Invitrogen), whereas BC-PAP cells were transfected using Lipo2000 (Invitrogen), according to manufacturers' instructions. For stable transfections all the cell lines were transfected with PATZ1-EGFP-C2 plasmid carrying human PATZ1 variant 4 cDNA, or with the empty vector pEGFP-C2 (Clontech). Stable transfectants were clonally selected in complete medium containing 1 μg/ml G418 (Life Technologies). pCEFL-HA [23 (link)], and HA-PATZ1 plasmids, carrying the human PATZ1 variant 4 cDNA fused to the HA tag, were used in the colony assay.

Chiappetta G., Valentino T., Vitiello M., Pasquinelli R., Monaco M., Palma G., Sepe R., Luciano A., Pallante P., Palmieri D., Aiello C., Rea D., Losito S.N., Arra C., Fusco A, & Fedele M. (2015). PATZ1 acts as a tumor suppressor in thyroid cancer via targeting p53-dependent genes involved in EMT and cell migration. Oncotarget, 6(7), 5310-5323.

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Cloning and Characterization of Trio and Piccolo

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The human Trio cDNA (accession number AF091395) in EGFP-C1 vector (Clontech) was provided by Dr. Anne Debant [42 (link)]. Myc-tagged Trio was generated by excising the GFP cassette with AgeI and BspEI and replacing it with a synthetic oligonucleotide encoding the Myc epitope. C-terminal truncated Trios were generated by using blunt end restriction endonucleases and ligating the resultant plasmid using the SmaI site in the EGFP backbone. These molecular biology reagents were purchased from New England Biolabs. Myc-tagged polypeptides encompassing various Spectrin repeats of Trio were generated by standard PCR cloning methodologies. The cDNA of full length (FL) Myc-Trio lacking amino acids 395–606 (Spectrin repeats #3 and #4) was generated using the gap overlap extension method of PCR. All PCR cloning was conducted with KOD DNA Polymerase (Novagen) and resultant cDNA products were sequenced to ensure fidelity.
The GFP-tagged rat Bassoon cDNA was a kind gift from Thomas Dresbach and Wilko Altrock [43 (link)]. To generate the cDNA encoding amino acids 4077–4875 of rat Piccolo (accession number NM_020098), we excised C-terminal PBH9/10-PDZ-C2A region Piccolo from the R2 plasmid [44 (link)] with EcoRI and ligated this insert into the EcoR1 site in EGFP-C2 (Clontech).

Terry-Lorenzo R.T., Torres V.I., Wagh D., Galaz J., Swanson S.K., Florens L., Washburn M.P., Waites C.L., Gundelfinger E.D., Reimer R.J, & Garner C.C. (2016). Trio, a Rho Family GEF, Interacts with the Presynaptic Active Zone Proteins Piccolo and Bassoon. PLoS ONE, 11(12), e0167535.

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Generation of shRNA and HA-tagged Mutant Constructs

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shRNA against μ2 and HA-μ2 plasmids resistant to shRNA μ2 were constructed as previously described24 (link). 60-mers oligonucleotides containing rat μ2 subunit cDNA target sequences (GTGGATGCCTTTCGCGTCA) of AP-2 were synthesized, annealed, and ligated into pSUPER vector according to manufacturer’s instruction. HA-μ2 construct was generated using Quick Change Site-Directed mutagenesis (Stratagene). The target sequence for shRNA μ2 was mutated to GTGGACGCTTTCCGGGTCA without the amino acid sequence change. EGFP-C2 is from clontech, GFP-Tau, VMAP2-GFP, GFP-synapsin-1, Na_v1.6-GFP was cloned as previously described52 (link). GCaMP6f from Addgene30 (link) is conjugated to synaptophysin.

Kyung J.W., Cho I.H., Lee S., Song W.K., Ryan T.A., Hoppa M.B, & Kim S.H. (2017). Adaptor Protein 2 (AP-2) complex is essential for functional axogenesis in hippocampal neurons. Scientific Reports, 7, 41620.

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Constructing pGFAP-CreERT2 Lentiviral Vector

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To construct pCDH-pGFAP-MCS-EF1-puro, the CMV promoter sequence of the pCDH-CMV-MCS-EF1-puro lentiviral vector (System Biosciences, USA) was removed by using two restriction enzymes, SnaBI and NheI. The pGFAP promoter sequence (−1,817 to −17 upstream from the pGFAP transcription start site +1) in the pGL3-basic-pGFAP promoter plasmid [8 (link)] was digested with EcoRI (blunted by the Klenow fragment) and NheI. Subsequently, the pGFAP promoter sequence was ligated into the aforementioned CMV promoter-deleted lentiviral vector. Finally, to construct a lentiviral vector containing the pGFAP-CreER^T2 gene construct, the CreER^T2 gene digested by EcoRI from the pCre-ER^T2 plasmid [19 (link)] was inserted into the identical restriction enzyme sites of pCDH-pGFAP-MCS-EF1-puro.
To generate the CMV-LoxP-EGFP-pA-LoxP construct, LCMV:EGFP(LoxP)MCS (No. 31377; Addgene, USA) was modified by removing the EGFP sequence by using NcoI and BsrGI. Then, the EGFP sequence from EGFP-C2 (No. 6083-1; Clontech Laboratories, USA) that had been digested with the same restriction enzymes was inserted into the aforementioned EGFP-deleted vector.

Hwang S.U., Eun K., Yoon J.D., Kim H, & Hyun S.H. (2018). Production of transgenic pigs using a pGFAP-CreERT2/EGFPLoxP inducible system for central nervous system disease models. Journal of Veterinary Science, 19(3), 434-445.

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