Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Jennifer Lei
Date created: 2008-10-19 11:00:00
Date modified: 2015-05-08 01:08:34
CMV-rtTA
| Types | DnaRegion |
| Roles | Regulatory promoter |
| Sequences | BBa_K080003_sequence (Version 1) |
Description
In our adapted version of the pTRIPZ tetracycline inducible, Tet-On system, we designed a constitutively active version of the transactivator, rtTA protein, under a CMV promoter, to control the expression of cardiac progenitor initiators, nkx-2.5 and gata4.rtTA
The pTRIPZ vector is engineered to be Tet-On. The Tet-On technology equips the pTRIPZ vector to provide for induced expression of a gene in the presence of doxycycline (www.clonetech.com). There are two main components on the pTRIPZ vector enabling induction: the tetracycline response element (TRE) and the transactivator. The TRE, modified from its natural state to consist of a string of operators fused to the CMV minimal promoter, exhibits reduced basal expression and tighter binding to the second component, the transactivator.
pTRIPZ transactivator, known as the reverse tetracycline transactivator 3 (rtTA3) binds to and activates expression from TRE promoters in the presence of doxycycline. The rtTA3 transactivator is a modified version of the wildtype in two ways. First, unlike the original tetracycline transactivator the rtTA3 is modified to bind to the TRE in the presence of doxycycline rather than in its absence. Secondly, there are three mutations within the transactivator that increase its sensitivity to doxycycline by 25 fold over the initial rtTA without increasing background activity (Das, et al. 2004).
Das, et al. (2004) "Viral evolution as a tool to improve the tetracycline-regulated gene expression system" JBC Vol 279: 18, 18776-18782.
Notes
CMV-rtTACla1 (6bp)-Xba1 (6bp) -cmv (646bp) ??? pac1 (8bp) ??? mlu1 (6bp) ??? kozak (6bp) ???rtTA (698bp)- Nhe1 (6bp) ??? sal1 (6bp) = 1388bp