Difference between revisions of "Part:BBa K4344028"

 
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<partinfo>BBa_K4344028 short</partinfo>
 
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The expression of the HSV-<i>UL19</i> 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 <span class="scientific-src">(Alekhina & Vassilenko, 2012)</span>. This isn’t always the first AUG, but the first AUG with a surrounding  Kozak-sequence <span class="scientific-src">(Dunston <i>et al.</i>,  2004)</span>. This sequence was missing in our plasmid initially.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4344028 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4344028 SequenceAndFeatures</partinfo>
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A missing Kozak-sequence was inserted to enhance the translation of HSV <i>UL19</i> AA816:1148-6xHis via PCR. The HSV <i>UL19</i> fragment was amplified with PCR using Phusion 2x Mastermix with HF-Buffer (NEB), 0.5 µM pRK5-Kozak-fwd., HindIII-HSV <i>UL19</i>-rev. and roughly 1 ng of pEX-A258-HSV-<i>UL19</i>-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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Revision as of 13:23, 12 October 2022


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.