Part:BBa_K2100002:Design
pENTR pEREx6
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 6
Illegal EcoRI site found at 18
Illegal EcoRI site found at 333
Illegal EcoRI site found at 343
Illegal XbaI site found at 42 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 6
Illegal EcoRI site found at 18
Illegal EcoRI site found at 333
Illegal EcoRI site found at 343 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 6
Illegal EcoRI site found at 18
Illegal EcoRI site found at 333
Illegal EcoRI site found at 343 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 6
Illegal EcoRI site found at 18
Illegal EcoRI site found at 333
Illegal EcoRI site found at 343
Illegal XbaI site found at 42 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 6
Illegal EcoRI site found at 18
Illegal EcoRI site found at 333
Illegal EcoRI site found at 343
Illegal XbaI site found at 42 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
ERE's are spaced within the locations of the TetO sites of the TRE inducible promoter. Additionally, since the tetO site is 19 base pairs and ERE is only 13 base pairs, we added 5 additional random and different base pairs upstream, directly in front of, the ERE sequence to maintain the geometry of the TRE promoter since the way the DNA folds is important and we didn't want the DNA to misfold by having an incorrect length. The ERE sequence has three unidentified base pairs in the middle of its sequence, which we chose to fill randomly and differently for each occurrence of an ERE site since there is no indication that they have any effect on the functionality of the binding to ER-alpha and also because IDT would not synthesize it with so many repeated sequences. We chose repetitions of 3,5, and 6 because the paper Klinge et al. [1] had most success with 3 synthetic EREs, then five was a middle number, and 6 is the number of TetO sites on TRE-tight that we wanted to mimic since that was a successful synthetically built inducible promoter. We additionally liked the fact that the synthetic pEREs drastically shortened the length of promoter from the 2000+ base pairs of the natural estrogen responsive promoter, pTFF1. We also decided to try to synthesize an inducible promoter rather than doing the fusion with VP16-GAL4 since we wanted to maintain and utilize as many natural components of the cell itself. [1]
This basic part entry vector is flanked by L4 and R1 sites, which are used to denote a promoter. This can be cascade with a gene (flanked by L1, L2 sites) using an LR reaction and cloning into a cloned into a backbone that has a negative selection marker between R4 and R2 sites. This part adheres to RFC 65 for recombination based cloning of mammalian parts.
Source
Synthesized by IDT. Sequence given b/n L4 R1 sites
References
[1]Sathya G, Li W, Klinge CM, Anolik JH, Hilf R, Bambara RA. Effects of multiple estrogen responsive elements, their spacing, and location on estrogen response of reporter genes. Mol Endocrinol. 1997;11(13):1994–2003.