Difference between revisions of "Part:BBa K337049"

Latest revision as of 17:56, 29 October 2010

pSMB_miMeasure

pSMB_miMeasure: basic scheme

pSMB_miMeasure is the standard fluorescent-based microRNA binding site characterization vector. It is composed of a mirrored construct setup. 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

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 2643
Illegal SpeI site found at 2657
Illegal PstI site found at 2680
12
INCOMPATIBLE 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
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 2643
Illegal BamHI site found at 257
Illegal XhoI site found at 2913
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 2643
Illegal SpeI site found at 2657
Illegal PstI site found at 2680
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 2643
Illegal SpeI site found at 2657
Illegal PstI site found at 2680
1000
COMPATIBLE WITH RFC[1000]

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 [[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 (BB-2, RFC12).
+pSMB_miMeasure is the standard fluorescent-based microRNA binding site characterization vector. It is composed of a mirrored construct setup. 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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