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Pbabe cova

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The PBabe-cOVA is a plasmid vector that can be used for the expression of proteins in mammalian cells. It contains a cytomegalovirus (CMV) promoter for high-level expression and an ovalbumin (cOVA) coding sequence.

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Lab products found in correlation

Pslik hygro, by Addgene (2 mentions) Tmem192, by OriGene (2 mentions) Hla a, by Addgene (2 mentions) Pbabe zeo, by Addgene (2 mentions) Gateway cloning system, by Thermo Fisher Scientific (2 mentions) Pci neo cova, by Addgene (2 mentions) Pinducer20, by Addgene (2 mentions) Nebuilder hifi dna assembly cloning kit, by New England Biolabs (2 mentions)

2 protocols using pbabe cova

1

Generation of Fluorescent Protein Constructs

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GFP-NBR1 and GFP-NBR1 dUBA were provided by Jayanta Debnath. LysoTag - TMEM192-mRFP-3xHA (TMRHA) - was generated by subcloning the cDNA of TMEM192 (Origene) together with monomeric Red Fluorescent Protein (mRFP) and 3xHA tag into the NheI and EcoRI sites of pLJM1 lentiviral vector. HLA-A-TurboID-FLAG (HLA-A-TrID) was generated by subcloning the cDNA of HLA-A (Addgene plasmid, #85162) into the EcoRI and NotI sites of the TurboID pLVX vector (gift from Roberto Zoncu, UC Berkeley). pMXs GFP-LC3-RFP was a gift from Noboru Mizushima (Addgene, plasmid #117413). For Dox-inducible expression of Atg4BC74A, mTurquoise2 or mStrawberry was fused to Atg4BC74A and inserted into either pSLIK-Hygro (used for in vitro studies) (Addgene, plasmid #25737) or pINDUCER20 (used for in vivo studies) (Addgene, plasmid #44012), using the Gateway Cloning system (Thermo Fisher Science). For the generation of OVA-expressing cells, the cOVA fragment was cloned from pCI-neo-cOVA (Addgene, plasmid #25097), fused with 2A peptide and mStrawberry sequences using NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs) according to manufacturer’s instruction, and inserted into the EcoRI and SalI sites of pBabe-zeo (Addgene, plasmid #1766) to generate pBabe-cOVA-2A-mStrawberry. Stable cOVA expression was confirmed and monitored with mStrawberry fluorescence.
Yamamoto K., Venida A., Yano J., Biancur D.E., Kakiuchi M., Gupta S., Sohn A.S., Mukhopadhyay S., Lin E.Y., Parker S.J., Banh R.S., Paulo J.A., Wen K.W., Debnath J., Kim G.E., Mancias J.D., Fearon D.T., Perera R.M, & Kimmelman A.C. (2020). Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I. Nature, 581(7806), 100-105.
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2

Generation of Fluorescent Protein Constructs

Check if the same lab product or an alternative is used in the 5 most similar protocols
GFP-NBR1 and GFP-NBR1 dUBA were provided by Jayanta Debnath. LysoTag - TMEM192-mRFP-3xHA (TMRHA) - was generated by subcloning the cDNA of TMEM192 (Origene) together with monomeric Red Fluorescent Protein (mRFP) and 3xHA tag into the NheI and EcoRI sites of pLJM1 lentiviral vector. HLA-A-TurboID-FLAG (HLA-A-TrID) was generated by subcloning the cDNA of HLA-A (Addgene plasmid, #85162) into the EcoRI and NotI sites of the TurboID pLVX vector (gift from Roberto Zoncu, UC Berkeley). pMXs GFP-LC3-RFP was a gift from Noboru Mizushima (Addgene, plasmid #117413). For Dox-inducible expression of Atg4BC74A, mTurquoise2 or mStrawberry was fused to Atg4BC74A and inserted into either pSLIK-Hygro (used for in vitro studies) (Addgene, plasmid #25737) or pINDUCER20 (used for in vivo studies) (Addgene, plasmid #44012), using the Gateway Cloning system (Thermo Fisher Science). For the generation of OVA-expressing cells, the cOVA fragment was cloned from pCI-neo-cOVA (Addgene, plasmid #25097), fused with 2A peptide and mStrawberry sequences using NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs) according to manufacturer’s instruction, and inserted into the EcoRI and SalI sites of pBabe-zeo (Addgene, plasmid #1766) to generate pBabe-cOVA-2A-mStrawberry. Stable cOVA expression was confirmed and monitored with mStrawberry fluorescence.
Yamamoto K., Venida A., Yano J., Biancur D.E., Kakiuchi M., Gupta S., Sohn A.S., Mukhopadhyay S., Lin E.Y., Parker S.J., Banh R.S., Paulo J.A., Wen K.W., Debnath J., Kim G.E., Mancias J.D., Fearon D.T., Perera R.M, & Kimmelman A.C. (2020). Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I. Nature, 581(7806), 100-105.
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