BBa_K203100 1 p31 pSMB_MEASURE: Promoter Measurement plasmid (mammalian) 2009-10-14T11:00:00Z 2015-05-08T01:11:22Z plasmid: modified from pcDNA5/FRT (Invitrogen) as described below GFP: PCR from a plasmid containing eGFP-N1 pSMB_MEASURE (SMB is for Synthetic Mammalian Biology) should be used for promoter characterization in mammalian cells. pSMB_MEASURE contains a reference promoter, JeT[1], 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. false false _301_ 0 5154 9 It's complicated true As a starting plasmid, we used pcDNA5/FRT (Invitrogen), as having this plasmid at hand was an immediate requirement for our project and we did not have the time to construct it from parts. We nevertheless highly modified it by performing two site-directed mutageneses to remove prohibited cutsites, we removed the CMV promoter it contained and inserted JeT, a well-described[1] synthetic promoter which we modified to contain a HindIII cutsite between the proximal and the core promoter for promoter screening. We also added full BBB_2 (Tom Knight) sites to it. Then, we synthesized it by Assembly PCR and inserted it via a MfeI and a PstI site. We then fixed eGFP plus a Kozak-sequence (eukaryotic RBS) into the plasmid backbone by cutting with PstI and BClI References: [1] Tornoe, J. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 297, 21-32 (2002). false Lars Velten, Simon Haas, Anne Rademacher, Hannah Meyer, Michael Bartoschek and Chenchen Zhu annotation2041656 1 JeT promoter range2041656 1 191 355 annotation2041652 1 pBR322_origin range2041652 1 3392 4011 annotation2041654 1 SV40 terminator range2041654 1 1117 1344 annotation2041650 1 Amp_R promoter range2041650 1 5068 5096 annotation2041653 1 Hygromycine Resistance Gene range2041653 1 1695 2702 annotation2041658 1 Kozak sequence range2041658 1 379 389 annotation2041648 1 GFP range2041648 1 387 1103 annotation2041657 1 BBB suffix range2041657 1 359 379 annotation2041651 1 Ampicillin Resistance Gene range2041651 1 4166 5026 annotation2041655 1 BBB prefix range2041655 1 171 191 annotation2041649 1 FRT-site range2041649 1 1628 1675 BBa_K203100_sequence 1 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igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 James Alastair McLaughlin Chris J. Myers 2017-03-06T15:00:00.000Z