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URL: http://www.addgene.org/102646
Proper Citation: RRID:Addgene_102646
Bacterial Resistance: Ampicillin
Defining Citation: PMID:
Vector Backbone Description: Backbone Marker:Markus Ralser (Addgene plasmid # 19407), Clontech = pTRE-T2; Vector Backbone:pTRE-Tight2; Vector Types:Mammalian Expression, RNAi; Bacterial Resistance:Ampicillin
Comments: In fusion cloning was used to insert a synthetic DNA template containing the mIR cassette from the pCDNA 6.2-GW/miR into the EcoRI and XbaI site of pTRE-T2-Tight. I engineered two BsaI sites into the miR cassette so users can digest the vector with BsaI for directional cloning of Blockit microRNAs using the Block-IT RNAi designer engine from Invitrogen. To make the BsaI sites unique I mutated out Bsa I sites in the original pTRE-T2 Tight vector and also removed XhoI and XbaI sites by site-directed mutagenesis from the pTRE-T2 vector and the corresponding puromycin/hygromycin resistance genes. I also dropped in a unique NheI and EagI restriction site between the ampicillin gene and the pMB101 origin of replication sequence so that the puromycin and hygromin genes could be placed into the pTRE-T2-miR vector with the infusion cloning method. Investigators can remove the miR cassette from the pTRE-T2 puromycin/hygromycin vector and using cloning methods to insert any cDNA of interest into the site for doxycline-inducible expression of the target cDNA. A single vector can be utilized from the production of tetracycline inducible miRNAs or cDNAs. For puromycin use .25 micrograms/ml to .50 micrograms/ml to get clones after a couple of weeks. Growth response curve tested in 293HEK cells. Suggest methylation deficient cells from NEB to alleviate methylation sensitive restriction sites like Xba I. However not recommended for long-term propagation and storage.
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Source: Addgene
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