Difference between revisions of "Part:BBa K337049"
| Line 3: | Line 3: | ||
[[Image:PSMB_miMeasure.png|thumb|250px|right|pSMB_miMeasure: basic scheme]] | [[Image:PSMB_miMeasure.png|thumb|250px|right|pSMB_miMeasure: basic scheme]] | ||
<br/> | <br/> | ||
| − | 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. | + | 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 pANY backbone. |
<br /> | <br /> | ||
Revision as of 06:11, 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 pANY backbone.
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]