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URL: http://www.addgene.org/177118
Proper Citation: RRID:Addgene_177118
Insert Name: MG1655 PlacZYA::Plac-gfp-muNS-kan
Bacterial Resistance: Kanamycin
Defining Citation: PMID:24361104
Vector Backbone Description: Vector Backbone:none; Vector Types:; Bacterial Resistance:Kanamycin
Comments: GFP-muNS expression is under the control of Plac promoter integrated into the endogenous lac locus. The native lac operon (lacZYA) is deleted. Kanamycin resistance casette is inserted downstream of the gfp-muNS gene for cloning purposes. Cloning method: Lambda RED recombination. The gfp-μNS fragment was amplified from plasmid pER12 (pBAD322A-gfp-μNS), which encodes an N-terminal GFP fusion to residues 471-721 of μNS (Broering et al., 2005) and then digested by KpnI/XhoI. The nptI gene was amplified from pKD4 plasmid and digested by XhoI/SmaI. Then, gfp-μNS and nptI gene fragments were triple ligated into the pKS plasmid. The resulting plasmid was used as a PCR template to generate a gfp-μNS-nptI fragment with flanking regions complementary to lacI and cynX, respectively. This fragment was then used to replace the lac operon (lacZYA) by one-step λ-recombination (Datsenko and Wanner, 2000). The resulting gfp-μNS-nptI replacement of lacZYA was transduced back into MG1655 by P1-phage to generate strain CJW4617. See Parry et al., 2014 for more details. This strain doesn't contain any plasmid.
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Source: Addgene
Data set from Kravitz, Owen and Kretizer's 2012 paper "Optogenetic identification of striatal projection neuron subtypes during in vivo recordings."
