Part:BBa_K4344028
Kozak Sequence (human RBS)
The expression of the HSV-UL19 mRNA was too low which is why we included an additional Kozak-sequence to our enhanced CMV-promoter. The Kozak-sequence is the conserved consensus sequence next to the promoter, surrounding the AUG for the start of transcription in eukaryotes. It plays a role in the binding of the ribosome complex to the AUG during initiation of transcription (Alekhina & Vassilenko, 2012). This isn’t always the first AUG, but the first AUG with a surrounding Kozak-sequence (Dunston et al., 2004). This sequence was missing in our plasmid initially.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
A missing Kozak-sequence was inserted to enhance the translation of HSV UL19 AA816:1148-6xHis via PCR. The HSV UL19 fragment was amplified with PCR using Phusion 2x Mastermix with HF-Buffer (NEB), 0.5 µM pRK5-Kozak-fwd., HindIII-HSV UL19-rev. and roughly 1 ng of pEX-A258-HSV-UL19-6xHis, the total reaction volume being 20 µL, for this purpose. The success of the PCR was evaluated on a 1.2 % TAE-Agarose gel stained with ethidium bromide. Successful PCR products were pooled and purified using the Qiagen PCR Clean-Up Kit. The concentration was measured using Nanodrop 2000.
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