Difference between revisions of "Part:BBa K203100"

Revision as of 09:57, 20 October 2009

pSMB_MEASURE: Promoter Measurement plasmid (mammalian)

Plasmid map of pSMB_MEASURE

pSMB_MEASURE (SMB is for Synthetic Mammalian Biology) should be used for promoter characterization in mammalian cells. pSMB_MEASURE contains a reference promoter, JeT (Part:BBa_K203112), which is flanked by BBb_2 (Tom Knight) sites and can therefore be replaced by the promoter to be measured. JeT is ideal as a reference promoter for a variety of reasons. First, it has an intermediate expression strength; second, it is regulated by a wide variety of transcription factors and low levels of change in fluorescence among different conditions. Third, we want to pay tribute to its creators as pioneers in synthetic promoter research.

We separated JeT's core promoter from its proximal promoter by a HindIII site; it can therefore be used for screening of synthetic proximal promoters or for modifiying the strength of a promoter by core promoter swapping (described in our wiki http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters ). In addition, it contains a FRT site which will allow for stable integration into mammalian cells also containing a FRT site. Thus, it provides the possibility to characterize the promoter in a defined genome and in this way helps to avoid some of the challenges we identified for promoter characterization. For the same reason, it also contains a mammalian selection marker (hygromycine). For the generation of the plasmid, please see our Notebook. As a reporter gene, it contains GFP, which is followed by a SV40 mammalian terminator. We generated another plasmid pSMB_REFERENCE, which contains mcherry instead of GFP. It can be used for normalizations to transfection efficiency.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 171
Illegal XbaI site found at 1655
Illegal SpeI site found at 185
Illegal PstI site found at 375
12
INCOMPATIBLE WITH RFC[12]
Illegal prefix found at 171
Plasmid lacks a suffix.
Illegal NheI site found at 361
Illegal PstI site found at 375
Illegal NotI site found at 367
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 171
Illegal BglII site found at 12
23
INCOMPATIBLE WITH RFC[23]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 171
Illegal XbaI site found at 1655
Illegal SpeI site found at 185
Illegal PstI site found at 375
25
INCOMPATIBLE WITH RFC[25]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 171
Illegal XbaI site found at 1655
Illegal SpeI site found at 185
Illegal PstI site found at 375
Illegal NgoMIV site found at 1515
Illegal NgoMIV site found at 2778
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI.rc site found at 4306
Illegal SapI site found at 3223

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K203100 short</partinfo>
-pSMB_MEASURE (SMB is for Synthetic Mammalian Biology) should be used for promoter characterization in mammalian cells. pSMB_MEASURE contains a reference promoter, JeT ([[Part:BBa_K203112]]), which is flanked by BBb_2 (Tom Knight) sites and can therefore be replaced by the promoter to be measured. JeT is ideal as a reference promoter for a variety of reasons. First, it has an intermediate expression strength; second, it is regulated by a wide variety of transcription factors and low levels of change in fluorescence among different conditions. Third, we want to pay tribute to its creators as pioneers in synthetic promoter research.
+[[Image:HD09_p31.png|thumb|left|none|Plasmid map of pSMB_MEASURE]] pSMB_MEASURE (SMB is for Synthetic Mammalian Biology) should be used for promoter characterization in mammalian cells. pSMB_MEASURE contains a reference promoter, JeT ([[Part:BBa_K203112]]), which is flanked by BBb_2 (Tom Knight) sites and can therefore be replaced by the promoter to be measured. JeT is ideal as a reference promoter for a variety of reasons. First, it has an intermediate expression strength; second, it is regulated by a wide variety of transcription factors and low levels of change in fluorescence among different conditions. Third, we want to pay tribute to its creators as pioneers in synthetic promoter research.
 We separated JeT's core promoter from its proximal promoter by a HindIII site; it can therefore be used for screening of synthetic proximal promoters or for modifiying the strength of a promoter by core promoter swapping (described in our wiki http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters ). In addition, it contains a FRT site which will allow for stable integration into mammalian cells also containing a FRT site. Thus, it provides the possibility to characterize the promoter in a defined genome and in this way helps to avoid some of the challenges we identified for promoter characterization. For the same reason, it also contains a mammalian selection marker (hygromycine). For the generation of the plasmid, please see our Notebook. As a reporter gene, it contains GFP, which is followed by a SV40 mammalian terminator. We generated another plasmid [[Part:BBa_K203099|pSMB_REFERENCE]], which contains mcherry instead of GFP. It can be used for normalizations to transfection efficiency.