Part:BBa_K3652006
Lentiviral Vector shRNA Regulatory Region (pLKO-tet-neo)
This part is the H1 promoter, tet-on operator system, and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). It can be the regulatory precursor for any shRNA sequence inserted into a generation 3 lentiviral cloning (transfer) plasmid, such as the tet-pLKO-neo plasmid which we inserted this part into. The human H1 promoter is a Pol III promoter commonly used for RNAi applications--specifically, as the promoter for siRNA or shRNA coding sequences. It is slightly weaker than the additionally common promoter, U6, with typically lower expression levels and shorter-term effects when compared on similar genes and shRNA sequences [1]. We used it preceding our shRNA sequence, because we wanted to avoid irreversible gene knockdown, or very long-term effects in our target users, as it could lead to excess angiogenesis, and harm the patients. Ultimately H1 is a good choice for those looking for a weaker knockdown than that typically seen with U6 promoters. The tet-on operator system is a very common regulatory system, developed in 1995 by Gossen et. al. It allows the activation of gene expression in the presence of tetracycline, or doxycycline via a variant of the TetR protein (developed by mutagenesis) [2]. This variant binds to tetO in the presence of tet/dox. Finally, the EMCV IRES was used as the viral ribosome binding site. Though not strictly necessary for successful transcription of the following shRNA/siRNA sequence, it has been shown to increase efficacy of expression of the shRNA [3], so we included it seeking more successful transcription of the shRNA, at least during the testing. If it validates the transformation of our plasmid, and produces high amounts of shRNA, its removable can be considered to alter the potency of the shRNA production.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 66
Illegal PstI site found at 274 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 274
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 66
Illegal PstI site found at 274 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 66
Illegal PstI site found at 274 - 1000COMPATIBLE WITH RFC[1000]
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