Pecfp c1

Manufactured by Takara Bio

Sourced in United States

The PECFP-C1 is a plasmid designed for the expression of enhanced cyan fluorescent protein (ECFP) in mammalian cells. It contains the ECFP coding sequence under the control of the CMV promoter for strong and constitutive expression.

Automatically generated - may contain errors

Lab products found in correlation

Peyfp c1, by Takara Bio (11 mentions) Pegfp c1, by Takara Bio (7 mentions) Pmcherry c1, by Takara Bio (6 mentions) Peyfp n1, by Takara Bio (6 mentions) Pecfp n1, by Takara Bio (4 mentions) Peyfp c1 expression vectors, by Takara Bio (3 mentions) Pcdna3, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Pegfp n1, by Takara Bio (2 mentions) On targetplus smartpool, by GE Healthcare (1 mentions) Pgex 2t, by GE Healthcare (1 mentions) Ptripz, by GE Healthcare (1 mentions) Quikchange xl site directed mutagenesis kit, by Agilent Technologies (1 mentions) Q5 polymerase, by New England Biolabs (1 mentions) Vent polymerase, by New England Biolabs (1 mentions) Pcmv tag2, by Agilent Technologies (1 mentions) Pirespuro3, by Takara Bio (1 mentions) Pflag cmv 2, by Merck Group (1 mentions) Pcw cas9, by Addgene (1 mentions) Pbind, by Promega (1 mentions)

Close spelling variants of this product

PECFP-C1 vector (8 citations) PECFP (6 citations)

31 protocols using pecfp c1

Subcloning GABARAP Genes for Fluorescent Fusion

Check if the same lab product or an alternative is used in the 5 most similar protocols

Genes for GABARAP, GABARAPL1 and GABARAPL2 were subcloned from GST-fusion plasmids (Addgene IDs 73948, 73945 and 73518) by PCR amplification into the XhoI and BamHI sites of peYFP-C1 or peCFP-C1(Clontech), yielding peYFP-C1/GABARAP, peYFP-C1/GABARAPL1 and peCFP-C1/GABARAPL2.

Simons I.M., Mohrlüder J., Feederle R., Kremmer E., Zobel T., Dobner J., Bleffert N., Hoffmann S, & Willbold D. (2019). The highly GABARAP specific rat monoclonal antibody 8H5 visualizes GABARAP in immunofluorescence imaging at endogenous levels. Scientific Reports, 9, 526.

+ Open protocol

+ Expand

Plasmid Construction and Knockdown Techniques

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Full-length or partial coding regions of each cDNA were amplified by PCR and cloned in-frame into the indicated vectors, including pEGFP-C1, pmCherry-C1, pEYFP-N1, pECFP-C1 (Takara Bio Inc.), pcDNA5/FRT/TO, pcDNA3.1+ (Invitrogen), pTRIPz (GE Healthcare), pGEX-2T (GE Healthcare), pET-14b (EMD Millipore), and pCGN (Fiordalisi et al., 2001 (link)). Amino acid substitutions were generated by site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis kit (Agilent Technologies). All plasmids were verified by bidirectional sequencing.
For siRNA knockdown, cells were transfected with ON-TARGETplus SMARTpools (GE Healthcare) targeting VPS35, PDE6D or nontargeting control using DharmaFECT 1 reagent according to the manufacturer’s instructions. For shRNA knockdown, targeting sequences for VPS35 were inserted into pTRIPz lentiviral vector by PCR, and lentiviral particles were generated with standard protocols (http://tcf.epfl.ch/files/content/sites/tcf/files/shared/LV_production.pdf). pTRIPz-based NRAS-targeting lentivirus vectors (Grabocka et al., 2014 (link)) were provided by D. Bar-Sagi (New York University Langone Medical Center, New York, NY). Stable cells lines were generated by lentiviral infection and selection with puromycin for 5 d. Knockdown was induced by 0.4 µg/ml doxycycline for at least 72 h.

Zhou M., Wiener H., Su W., Zhou Y., Liot C., Ahearn I., Hancock J.F, & Philips M.R. (2016). VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking. The Journal of Cell Biology, 214(4), 445-458.

+ Open protocol

+ Expand

Fluorescent Protein Constructs for Cell and Nuclear Imaging

Check if the same lab product or an alternative is used in the 5 most similar protocols

Construct expressing Tks5^GFP was a kind gift of Dr S. Courtneidge (OHSU, Portland, OR). Retroviral vector encoding H2B^GFP and ^GFPLMNA were provided by F. A. Dick (UWO, London, ON, Canada) and T. Misteli (Addgene #17662), respectively. Lentiviral vector encoding H2B^mCh was from M. Mercola (Addgene #21217). The ^GFPCentrin-1 plasmid was obtained from M. Bornens (Institut Curie, Paris, France). pRK5^mycRac1L61 was a kind gift of Dr S. Etienne-Manneville (Institut Pasteur, Paris, France). ^GFPDN-KASH and ^GFPKASHext were generated by inserting the C-terminal domain of Nesprin-2 (corresponding to the last 331 amino acids) into the XhoI–BamHI site of pECFP-C1 (Takara Bio Inc.). KASHext was derived from the DN-KASH construct by adding a C-terminal VDGTAGPGSTGSR amino acid extension^{29 (link)}. For Lis1 rescue experiment, LIS1^GFP construct was mutagenized to make it resistant to siLIS1#06 siRNA using the QuikChange Primer Design kit (Agilent) using: forward primer; 5′-CTCTGCTTCcGAGGATGCTACTATcAAaGTtTGGGAcTACGAGACTGGAGATTTTGAA-3′; reverse primer: 5′-AAAATCTCCAGTCTCGTAgTCCCAaACtTTgATAGTAGCATCCTCgGAAGCAGAG-3′.

Infante E., Castagnino A., Ferrari R., Monteiro P., Agüera-González S., Paul-Gilloteaux P., Domingues M.J., Maiuri P., Raab M., Shanahan C.M., Baffet A., Piel M., Gomes E.R, & Chavrier P. (2018). LINC complex-Lis1 interplay controls MT1-MMP matrix digest-on-demand response for confined tumor cell migration. Nature Communications, 9, 2443.

+ Open protocol

+ Expand

STIM1 Mutation Expression Constructs

Check if the same lab product or an alternative is used in the 5 most similar protocols

STIM1 DNA constructs (accession number NM_003156), with specific point mutations pre-inserted using the QuikChange site-directed mutagenesis kit (StrataGene, San Diego, CA, USA), were cloned into pECFP-C1 and pEYFP-C1 expression vector systems (Clontech, now Takara, Mountain View, CA, USA). Successful mutation and vector-insertion was confirmed by sequence analysis (Eurofins Genomics, Vienna, Austria).

Sallinger M., Tiffner A., Schmidt T., Bonhenry D., Waldherr L., Frischauf I., Lunz V., Derler I., Schober R, & Schindl R. (2020). Luminal STIM1 Mutants that Cause Tubular Aggregate Myopathy Promote Autophagic Processes. International Journal of Molecular Sciences, 21(12), 4410.

+ Open protocol

+ Expand

Cloning and Expression of TRIB1

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cloning was performed using standard molecular biology tools; olignucleotides are listed in the S1 Supplementary Materials section. TRIB1 was amplified from a construct obtained from Origene (pCMV6-XL5TRIB1). The open reading frame was amplified by PCR using Vent polymerase (New England BioLabs) and transferred in either pLVX for lentiviral expression or in either pCMV-Tag2 (Agilent) or pECFP-C1 (Clontech) for transient transfection of proteins containing N-terminal FLAG tag or eCFP tags, respectively. Deletions were performed using the Q5 polymerase (New England BioLabs). All constructs were verified by Sanger sequencing.

Soubeyrand S., Martinuk A., Lau P, & McPherson R. (2016). TRIB1 Is Regulated Post-Transcriptionally by Proteasomal and Non-Proteasomal Pathways. PLoS ONE, 11(3), e0152346.

+ Open protocol

+ Expand

Plasmid Transfection and Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Unless stated otherwise, reagents were obtained from Sigma (St. Louis, MO, USA). Lipofectamine 2000 and enzymes used for modifying DNA were purchased from Invitrogen (Carlsbad, CA, USA). The primers for cloning were ordered from IDT (Coralville, IA, USA). Plasmids pECFP-C1 and pIRESpuro3 were purchased from Clontech (Mountain View, CA, USA).

Kolossov V.L., Hanafin W.P., Beaudoin J.N., Bica D.E., DiLiberto S.J., Kenis P.J, & Gaskins H.R. (2014). Inhibition of glutathione synthesis distinctly alters mitochondrial and cytosolic redox poise. Experimental biology and medicine (Maywood, N.J.), 239(4), 394-403.

+ Open protocol

+ Expand

Plasmid Construction for Estrogen Receptor and YAP1 Study

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

pCW-FLAG-WT, Y537S, or D538G were created from pCW-Cas9 [62 (link)] (Addgene). pFLAG-CMV constructs were made from pFLAG-CMV2 (Sigma). YFP-tagged ESR1-YAP1 was constructed by PCR of the ESR1-YAP1 open reading frame (ORF) [11 (link)], followed by ligation into digested pECFP-C1 (Clontech). pBIND-YAP1 (GAL4 DBD-YAP1 amino acid 230–504 fusion expression plasmid) was constructed by ligation into pBIND (Promega). All constructs were sequenced.

Gates L.A., Gu G., Chen Y., Rohira A.D., Lei J.T., Hamilton R.A., Yu Y., Lonard D.M., Wang J., Wang S.P., Edwards D.G., Lavere P.F., Shao J., Yi P., Jain A., Jung S.Y., Malovannaya A., Li S., Shao J., Roeder R.G., Ellis M.J., Qin J., Fuqua S.A., O’Malley B.W, & Foulds C.E. (2018). Proteomic profiling identifies key coactivators utilized by mutant ERα proteins as potential new therapeutic targets. Oncogene, 37(33), 4581-4598.

+ Open protocol

+ Expand

Orai1 Construct Cloning and Mutation

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

N-terminally tagged Orai1 constructs (accession number NM_032790.3) were cloned into the SalI and SmaI restriction sites of pECFP-C1 and pEYFP-C1 expression vectors (Clontech). GFP-TFEB and GFP-MITF were purchased from Addgene and subcloned into peCFP vectors. hOrai1 Δ1-64, N223A, and cysteine-free (C126V, C143V, C195V) constructs were used for crosslinking as described in (12 (link)). Point mutations in Orai1 constructs were introduced using the QuikChange site-directed mutagenesis kit (Stratagene). The integrity of all resulting mutants was confirmed by sequence analysis (Eurofins Genomics).

Frischauf I., Litviňuková M., Schober R., Zayats V., Svobodová B., Bonhenry D., Lunz V., Cappello S., Tociu L., Reha D., Stallinger A., Hochreiter A., Pammer T., Butorac C., Muik M., Groschner K., Bogeski I., Ettrich R.H., Romanin C, & Schindl R. (2017). Transmembrane helix connectivity in Orai1 controls two gates for calcium-dependent transcription. Science signaling, 10(507), eaao0358.

+ Open protocol

+ Expand

Molecular Cloning of STIM1 and Orai1

Check if the same lab product or an alternative is used in the 5 most similar protocols

Full-length complementary DNA of human STIM1 was inserted into pECFP-C1, pEGFP-C1 and pEYFP-C1 vectors (Clontech) between BglII and EcoRI sites to generate STIM1-CFP, STIM1-GFP and STIM1-YFP. The sequence of SOAR was amplified by PCR and cloned into pCMV-Myc (Clontech) vector between EcoRI and BglII sites to generate the Myc-SOAR. Full-length cDNA of human Orai1 was inserted into pmCherry-C1 vector (Clontech) between BglII and EcoRI sites to generate mCherry-Orai1. Full-length cDNA of human Orai1 was inserted into pCMV-Myc vector between EcoRI and BglII sites to generate Myc-Orai1. GFP-STIM1 truncations were generated by introducing stop codon at the specific position. Full-length cDNA of rat CaM was cloned into pET-33(b) vector (Novagen) between NcoI and XhoI. mCherry-Orai1, SOAR-GFP, myc-Orai1, myc-SOAR and MBP-SOAR have been previously described^{30 (link)}. Mutations were generated by using the Quick-change mutagenesis kit (Agilent). Primers used in this study were listed in Supplementary Table 1.

Li X., Wu G., Yang Y., Fu S., Liu X., Kang H., Yang X., Su X.C, & Shen Y. (2017). Calmodulin dissociates the STIM1-Orai1 complex and STIM1 oligomers. Nature Communications, 8, 1042.

+ Open protocol

+ Expand

Orai1 and STIM1 Fluorescent Constructs

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human Orai1 (Orai1; accession no. NM_032790) was provided by A. Rao’s Laboratory, Harvard Medical School. N-terminally tagged Orai1 constructs were cloned with Sal I and Sma I restriction sites of pECFP-C1 and pEYFP-C1 expression vectors (Clontech). pEYFP-Orai1 served as template for the generation of pEYFP–Orai1 L74I and pEYFP–Orai1 Y80S. All mutants were constructed with the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The integrity of all resulting clones was confirmed by sequence analysis. Human STIM1 (STIM1; accession no. NM_003156) N-terminally tagged with enhanced CFP (ECFP) and enhanced yellow fluorescent protein (EYFP) was provided by T. Meyer’s Laboratory, Stanford University.

Derler I., Jardin I., Stathopulos P.B., Muik M., Fahrner M., Zayats V., Pandey S.K., Poteser M., Lackner B., Absolonova M., Schindl R., Groschner K., Ettrich R., Ikura M, & Romanin C. (2016). Cholesterol modulates Orai1 channel function. Science signaling, 9(412), ra10.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!