Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Lars Velten, Simon Haas, Anne Rademacher, Hannah Meyer, Michael Bartoschek and Chenchen Zhu
Date created: 2009-10-14 11:00:00
Date modified: 2015-05-08 01:11:22
pSMB_MEASURE: Promoter Measurement plasmid (mammalian)
| Types | DnaRegion |
| Roles | plasmid_vector Plasmid_Backbone |
| Sequences | BBa_K203100_sequence (Version 1) |
Description
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.
Notes
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 BClIReferences: [1] Tornoe, J. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 297, 21-32 (2002).
Source
plasmid: modified from pcDNA5/FRT (Invitrogen) as described belowGFP: PCR from a plasmid containing eGFP-N1
| Sequence Annotation | Location | Component / Role(s) |
| GFP FRT-site Amp_R promoter Ampicillin Resistance Gene pBR322_origin Hygromycine Resistance Gene SV40 terminator BBB prefix JeT promoter BBB suffix Kozak sequence | 387,1103 1628,1675 5068,5096 4166,5026 3392,4011 1695,2702 1117,1344 171,191 191,355 359,379 379,389 | CDS feature/protein feature/misc sequence_feature feature/promoter promoter CDS feature/protein sequence_feature feature/misc sequence_feature feature/misc polyA_site feature/polya feature/dna sequence_feature feature/promoter promoter feature/dna sequence_feature feature/rbs ribosome_entry_site |