Difference between revisions of "Part:BBa K1890020"
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This part is based on the part <partinfo>BBa_E0840</partinfo>, which already contains RBS, GFP gene and terminators. By means of PCR we added the strong constitutive promoter <partinfo>BBa_J23100</partinfo>. Two PCR reactions were performed with the following primers.
 
This part is based on the part <partinfo>BBa_E0840</partinfo>, which already contains RBS, GFP gene and terminators. By means of PCR we added the strong constitutive promoter <partinfo>BBa_J23100</partinfo>. Two PCR reactions were performed with the following primers.
 
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                                                <caption><b>Table 1:</b> Primers used to add promoter to GFP biobrick</caption>
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                                                <th>Primer name</th>
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                                                <th>Sequence</th>
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                                                    <tr>
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    <caption><b>Table 1:</b>Primers used for construction of this part.</caption>
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                                                        <td>E0840_FW</td>
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                                                        <td>ATTAAAGAGGAGAAATACTAGATGCGTAAAGG</td>
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    <th class="tg-8xqh">Primer name</th>
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                                                    </tr>
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    <th class="tg-5mgg">Sequence</th>
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                                                        <td>J23100-E0840_FW</td>
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                                                        <td>CGGCGAATTCGCGGCCGCTTCTAGAGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCATTAAAGAGGAGAAATACTAGATGCGTAAAGG</td>
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    <td class="tg-yw4l">E0840_FW</td>
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                                                    </tr>
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    <td class="tg-yw4l">ATTAAAGAGGAGAAATACTAGATGCGTAAAGG</td>
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                                                        <td>VR</td>
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                                                        <td>ATTACCGCCTTTGAGTGAGC</td>
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    <td class="tg-yw4l">J23100-E0840_FW</td>
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                                                    </tr>  
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    <td class="tg-yw4l">CGGCGAATTCGCGGCCGCTTCTAGAGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCATTAAAGAGGAGAAATACTAGATGCGTAAAGG</td>
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    <td class="tg-yw4l">VR</td>
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<figure>
 
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<img src="https://static.igem.org/mediawiki/2016/4/45/T--TU_Delft--BBa_K1890020_construction.png" width="80%">
 
<img src="https://static.igem.org/mediawiki/2016/4/45/T--TU_Delft--BBa_K1890020_construction.png" width="80%">

Revision as of 15:06, 9 October 2016


GFP with strong constitutive promoter, RBS and terminator

Indroduction

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria. This mutant represents a series of mutations resulting in enhanced maturation and emission [1]. It is expressed under control of the strong constitutive promoter BBa_J23100, strong RBS BBa_B0030 and terminators BBa_B0010 and BBa_B0012.

Construction

This part is based on the part BBa_E0840, which already contains RBS, GFP gene and terminators. By means of PCR we added the strong constitutive promoter BBa_J23100. Two PCR reactions were performed with the following primers.

Table 1: Primers used to add promoter to GFP biobrick
Primer name Sequence
E0840_FW ATTAAAGAGGAGAAATACTAGATGCGTAAAGG
J23100-E0840_FW CGGCGAATTCGCGGCCGCTTCTAGAGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCATTAAAGAGGAGAAATACTAGATGCGTAAAGG
VR ATTACCGCCTTTGAGTGAGC
Figure 1: Construction of the biobrick K1890020 by means of two PCRs, using biobrick E0840 as a template.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 700

References

[1] Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1), 33-38.