Difference between revisions of "Part:BBa K337049"
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[[Image:PSMB_miMeasure.png|thumb|250px|right|pSMB_miMeasure: basic scheme]] | [[Image:PSMB_miMeasure.png|thumb|250px|right|pSMB_miMeasure: basic scheme]] | ||
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| − | pSMB_miMeasure is the standard fluorescent-based microRNA binding site characterization vector. It is composed of a mirrored construct setup (fig 1). Two fluorescent proteins EGFP and EBFP2 are driven by a bidirectional CMV promoter to ensure the same expression strength at both sides. The two fluorescent proteins are destabilized by fusion to a MODC degradation domain which leads to a protein half life of two hours. The shortened half life of the destabilized fluorescent proteins enables for time-laps experiments in order to analyze binding site properties in a dynamic microRNA environment. Furthermore the plasmid contains a BBb_2 (RFC12, RFC41) standard site behind the EGFP to enable user friendly exchange of binding site of choice. BamHI and HindIII sites behind the EBFP2 enable for cloning in a reference binding site. The plasmid further contains an ampicillin resistance for selection in bacteria. As the complete Plasmid has been synthesized by Geneart, it is in the pMA backbone. | + | pSMB_miMeasure is the standard fluorescent-based microRNA binding site characterization vector. It is composed of a mirrored construct setup (fig 1). Two fluorescent proteins EGFP and EBFP2 are driven by a bidirectional CMV promoter to ensure the same expression strength at both sides. The two fluorescent proteins are destabilized by fusion to a MODC degradation domain which leads to a protein half life of two hours. The shortened half life of the destabilized fluorescent proteins enables for time-laps experiments in order to analyze binding site properties in a dynamic microRNA environment. Furthermore the plasmid contains a BBb_2 (RFC12, RFC41) standard site behind the EGFP to enable user friendly exchange of binding site of choice. BamHI and HindIII sites behind the EBFP2 enable for cloning in a reference binding site. The plasmid further contains an ampicillin resistance for selection in bacteria. As the complete Plasmid has been synthesized by Geneart, it is in the pMA backbone (BB-2, RFC12). |
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Revision as of 09:32, 27 October 2010
pSMB_miMeasure
pSMB_miMeasure is the standard fluorescent-based microRNA binding site characterization vector. It is composed of a mirrored construct setup (fig 1). Two fluorescent proteins EGFP and EBFP2 are driven by a bidirectional CMV promoter to ensure the same expression strength at both sides. The two fluorescent proteins are destabilized by fusion to a MODC degradation domain which leads to a protein half life of two hours. The shortened half life of the destabilized fluorescent proteins enables for time-laps experiments in order to analyze binding site properties in a dynamic microRNA environment. Furthermore the plasmid contains a BBb_2 (RFC12, RFC41) standard site behind the EGFP to enable user friendly exchange of binding site of choice. BamHI and HindIII sites behind the EBFP2 enable for cloning in a reference binding site. The plasmid further contains an ampicillin resistance for selection in bacteria. As the complete Plasmid has been synthesized by Geneart, it is in the pMA backbone (BB-2, RFC12).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2643
Illegal SpeI site found at 2657
Illegal PstI site found at 2680 - 12INCOMPATIBLE WITH RFC[12]Illegal prefix found in sequence at 2643
Illegal NheI site found at 2665
Illegal PstI site found at 2680
Illegal NotI site found at 2673 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2643
Illegal BamHI site found at 257
Illegal XhoI site found at 2913 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2643
Illegal SpeI site found at 2657
Illegal PstI site found at 2680 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2643
Illegal SpeI site found at 2657
Illegal PstI site found at 2680 - 1000COMPATIBLE WITH RFC[1000]