pTRE-T2-miR-PURO
(Plasmid #102646)

• Purpose: (Empty Backbone) Tet inducible microRNA and cDNA expression
• Depositing Lab: Michael E. Wright
• Publication: Wright lab backbones (unpublished)

Backbone

• Vector backbone: pTRE-Tight2
• Backbone manufacturer: Markus Ralser (Addgene plasmid # 19407), Clontech = pTRE-T2
• Vector type: Mammalian Expression, RNAi
• Selectable markers: Puromycin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: None

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor 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.