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Team:Warwick/Parts
From 2014.igem.org
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<b>NF-kappaB repressing factor (Homo sapiens)</b> | <b>NF-kappaB repressing factor (Homo sapiens)</b> | ||
<p>This IRES (internal ribosome entry site) is derived from the long 5’ untranslated region of the NF-kB repressing factor, a multifunctional cytokine interferon-β. This forms a secondary structure, shown below with sequence, which directs ribosomes to the downstream start codon by a cap-dependent mechanism. Following experimentation this was shown to have a 30-fold higher than picornaviral IRESs (Oumard, 2000). This was compared to the EMCV IRES and the poliovirus IRES in HeLa cells, murine embryonic stem cells and embryonic fibroblasts, using the firefly luciferase. The level of fluorescence seen was 92-fold higher than EMCV and 130-fold more active than the poliovirus IRES. NKRF acts in a distance independent manner and has a very high efficiency of translation initiation.</p> | <p>This IRES (internal ribosome entry site) is derived from the long 5’ untranslated region of the NF-kB repressing factor, a multifunctional cytokine interferon-β. This forms a secondary structure, shown below with sequence, which directs ribosomes to the downstream start codon by a cap-dependent mechanism. Following experimentation this was shown to have a 30-fold higher than picornaviral IRESs (Oumard, 2000). This was compared to the EMCV IRES and the poliovirus IRES in HeLa cells, murine embryonic stem cells and embryonic fibroblasts, using the firefly luciferase. The level of fluorescence seen was 92-fold higher than EMCV and 130-fold more active than the poliovirus IRES. NKRF acts in a distance independent manner and has a very high efficiency of translation initiation.</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2014/7/74/Warwick_Sequence_image_1.jpg | + | |
| - | <p><font color ="blue" size="1pt">Figure 1: Sequence and seconary structure of the NRF 5'UTR (EMBL accession number AJO11812) Oumard, 2000</font></p> | + | <div style="text-align: center"><img src="https://static.igem.org/mediawiki/2014/7/74/Warwick_Sequence_image_1.jpg"></div> |
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| + | <p><font color ="blue" size="1pt"><div style="text-align: center">Figure 1: Sequence and seconary structure of the NRF 5'UTR (EMBL accession number AJO11812) Oumard, 2000</div></font></p> | ||
<p>The sequence seen in Figure 1 is the one we used. This was cloned with a green fluorescent protein (iGEM part BBa_E0040) which we codon optimised for use in human cells and compared with a similar construct using EMCV IRES in place of the NKRF. We transfected the RNA of these constructs into Huh 7.5 cells in which, to our knowledge, the NKRF IRES has not been tested.</p> | <p>The sequence seen in Figure 1 is the one we used. This was cloned with a green fluorescent protein (iGEM part BBa_E0040) which we codon optimised for use in human cells and compared with a similar construct using EMCV IRES in place of the NKRF. We transfected the RNA of these constructs into Huh 7.5 cells in which, to our knowledge, the NKRF IRES has not been tested.</p> | ||
Revision as of 14:01, 29 August 2014
PARTS
IRES comparison
NF-kappaB repressing factor (Homo sapiens)This IRES (internal ribosome entry site) is derived from the long 5’ untranslated region of the NF-kB repressing factor, a multifunctional cytokine interferon-β. This forms a secondary structure, shown below with sequence, which directs ribosomes to the downstream start codon by a cap-dependent mechanism. Following experimentation this was shown to have a 30-fold higher than picornaviral IRESs (Oumard, 2000). This was compared to the EMCV IRES and the poliovirus IRES in HeLa cells, murine embryonic stem cells and embryonic fibroblasts, using the firefly luciferase. The level of fluorescence seen was 92-fold higher than EMCV and 130-fold more active than the poliovirus IRES. NKRF acts in a distance independent manner and has a very high efficiency of translation initiation.
The sequence seen in Figure 1 is the one we used. This was cloned with a green fluorescent protein (iGEM part BBa_E0040) which we codon optimised for use in human cells and compared with a similar construct using EMCV IRES in place of the NKRF. We transfected the RNA of these constructs into Huh 7.5 cells in which, to our knowledge, the NKRF IRES has not been tested.
