Plasmid Preparation:Article Title: Multi-input chemical control of protein dimerization for programming graded cellular responses.
Article Snippet: .. These vectors expressed only a tagBFP downstream of the CMV, and the guide plus 2x MS2 (wt + f6 sequences) under the U6 promoter. pCDNA5/FRT/TO-Lifeact-mCherry was created from mCherry-Lifeact-7, a gift from Michael Davidson (Addgene plasmid 54491). pEF5-FRT-mCherry-NS3aCAAX-IRES-EGFP-DNCR2-P2a-BFP-GNCR1 was created by assembling readers and fluorescent proteins from other constructs in a pEF5-FRT backbone obtained by digestion of Addgene plasmid 61684, a gift from Maxence Nachury. pPB-NS3aCAAX-IRES-EGFP-DNCR2-TIAM-P2a-BFP-GNCR1-LARG and the pEF5 version of the same construct, as well as NS3a-CAAX-IRES-EGFP-DNCR2-TIAM-P2aBFP-GNCR1 and NS3a-CAAX-IRES-EGFP-DNCR2-P2a-BFP-GNCR1-LARG, were assembled with NS3a, reader and fluorescent protein fragments from the previously mentioned construct, with addition of signaling effector domains from the following sources: human TIAM DH-domain residues 1033–1240 from a Maly Lab source and LARG DH-domain, which was a gift from Michael Glotzer (Addgene plasmid 80408). .. The PiggyBac vector used for these two constructs was linearized by digesting the multiple cloning site of PB501B (Systems Biosciences) with EcoRI and BamHI (New England BioLabs). pLenti-UAS-minCMV-mCherry/CMV-Gal4DBD-NS3a-P2a-DNCR2-VPR was based on a pLenti-UAS-minCMV-mCherry/CMV-Gal4DBD-ERT2VP16 vector, a gift from Kenneth Matreyek (from which the Gal4-UAS-minCMV was from Addgene plasmid 79130, a gift from Wendell Lim), which was digested with BamHI-HF and SexA1 to insert the NS3a-P2a-DNCR2-VPR fragment.
Article Title: Multi-input chemical control of protein dimerization for programming graded cellular responses
Article Snippet: .. These vectors expressed only a tagBFP downstream of the CMV, and the guide plus 2x MS2 (wt + f6 sequences) under the U6 promoter. pCDNA5/FRT/TO-Lifeact-mCherry was created from mCherry-Lifeact-7, a gift from Michael Davidson (Addgene plasmid 54491). pEF5-FRT-mCherry-NS3a-CAAX-IRES-EGFP-DNCR2-P2a-BFP-GNCR1 was created by assembling readers and fluorescent proteins from other constructs in a pEF5-FRT backbone obtained by digestion of Addgene plasmid 61684, a gift from Maxence Nachury. pPB-NS3a-CAAX-IRES-EGFP-DNCR2-TIAM-P2a-BFP-GNCR1-LARG and the pEF5 version of the same construct, as well as NS3a-CAAX-IRES-EGFP-DNCR2-TIAM-P2a-BFP-GNCR1 and NS3a-CAAX-IRES-EGFP-DNCR2-P2a-BFP-GNCR1-LARG and were assembled with NS3a, reader, and fluorescent protein fragments from the previously mentioned construct, with addition of signaling effector domains from the following sources: human TIAM DH-domain residues 1033–1240 from a Maly lab source, and LARG DH-domain was a gift from Michael Glotzer (Addgene plasmid 80408). .. The PiggyBac vector used for these two constructs was linearized by digesting the multiple cloning site of PB501B (Systems Biosciences) with EcoRI and BamHI (New England BioLabs). pLenti-UAS-minCMV-mCherry/CMV-Gal4DBD-NS3a-P2a-DNCR2-VPR was based on a pLenti-UAS-minCMV-mCherry/CMV-Gal4DBD-ERT2VP16 vector, a gift from Kenneth Matreyek, (from which the Gal4-UAS-minCMV was from Addgene plasmid 79130, a gift from Wendell Lim) which was digested with BamHI-HF and SexA1 to insert the NS3a-P2a-DNCR2-VPR fragment.
Construct:Article Title: Multi-input chemical control of protein dimerization for programming graded cellular responses.
Article Snippet: .. These vectors expressed only a tagBFP downstream of the CMV, and the guide plus 2x MS2 (wt + f6 sequences) under the U6 promoter. pCDNA5/FRT/TO-Lifeact-mCherry was created from mCherry-Lifeact-7, a gift from Michael Davidson (Addgene plasmid 54491). pEF5-FRT-mCherry-NS3aCAAX-IRES-EGFP-DNCR2-P2a-BFP-GNCR1 was created by assembling readers and fluorescent proteins from other constructs in a pEF5-FRT backbone obtained by digestion of Addgene plasmid 61684, a gift from Maxence Nachury. pPB-NS3aCAAX-IRES-EGFP-DNCR2-TIAM-P2a-BFP-GNCR1-LARG and the pEF5 version of the same construct, as well as NS3a-CAAX-IRES-EGFP-DNCR2-TIAM-P2aBFP-GNCR1 and NS3a-CAAX-IRES-EGFP-DNCR2-P2a-BFP-GNCR1-LARG, were assembled with NS3a, reader and fluorescent protein fragments from the previously mentioned construct, with addition of signaling effector domains from the following sources: human TIAM DH-domain residues 1033–1240 from a Maly Lab source and LARG DH-domain, which was a gift from Michael Glotzer (Addgene plasmid 80408). .. The PiggyBac vector used for these two constructs was linearized by digesting the multiple cloning site of PB501B (Systems Biosciences) with EcoRI and BamHI (New England BioLabs). pLenti-UAS-minCMV-mCherry/CMV-Gal4DBD-NS3a-P2a-DNCR2-VPR was based on a pLenti-UAS-minCMV-mCherry/CMV-Gal4DBD-ERT2VP16 vector, a gift from Kenneth Matreyek (from which the Gal4-UAS-minCMV was from Addgene plasmid 79130, a gift from Wendell Lim), which was digested with BamHI-HF and SexA1 to insert the NS3a-P2a-DNCR2-VPR fragment.
Article Title: Multi-input chemical control of protein dimerization for programming graded cellular responses
Article Snippet: .. These vectors expressed only a tagBFP downstream of the CMV, and the guide plus 2x MS2 (wt + f6 sequences) under the U6 promoter. pCDNA5/FRT/TO-Lifeact-mCherry was created from mCherry-Lifeact-7, a gift from Michael Davidson (Addgene plasmid 54491). pEF5-FRT-mCherry-NS3a-CAAX-IRES-EGFP-DNCR2-P2a-BFP-GNCR1 was created by assembling readers and fluorescent proteins from other constructs in a pEF5-FRT backbone obtained by digestion of Addgene plasmid 61684, a gift from Maxence Nachury. pPB-NS3a-CAAX-IRES-EGFP-DNCR2-TIAM-P2a-BFP-GNCR1-LARG and the pEF5 version of the same construct, as well as NS3a-CAAX-IRES-EGFP-DNCR2-TIAM-P2a-BFP-GNCR1 and NS3a-CAAX-IRES-EGFP-DNCR2-P2a-BFP-GNCR1-LARG and were assembled with NS3a, reader, and fluorescent protein fragments from the previously mentioned construct, with addition of signaling effector domains from the following sources: human TIAM DH-domain residues 1033–1240 from a Maly lab source, and LARG DH-domain was a gift from Michael Glotzer (Addgene plasmid 80408). .. The PiggyBac vector used for these two constructs was linearized by digesting the multiple cloning site of PB501B (Systems Biosciences) with EcoRI and BamHI (New England BioLabs). pLenti-UAS-minCMV-mCherry/CMV-Gal4DBD-NS3a-P2a-DNCR2-VPR was based on a pLenti-UAS-minCMV-mCherry/CMV-Gal4DBD-ERT2VP16 vector, a gift from Kenneth Matreyek, (from which the Gal4-UAS-minCMV was from Addgene plasmid 79130, a gift from Wendell Lim) which was digested with BamHI-HF and SexA1 to insert the NS3a-P2a-DNCR2-VPR fragment.
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