Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Kaj Bernhardt
Date created: 2009-10-16 11:00:00
Date modified: 2015-05-08 01:11:42
P_F3_ZeoR_F3_RFP. [FLP]->RFP
| Types | DnaRegion |
| Roles | Reporter engineered_region |
| Sequences | BBa_K260017_sequence (Version 1) |
Description
This is a FLP recombinase reporter. The gene for RFP (mRFP1) has no Start-codon and is silenced by the upstream terminator. If FLP recombinase levels are high enough, recombination between both F3 sites will delete the intervening ZeoR gene plus terminator. Now RFP comes under the control of the constitutive promoter that was driving expression of an F3-ZeoR fusion protein before. The result is an F3-RFP fusion protein that turns the cell red.Since this part is available in plasmid backbones pTetFlp (BBa_K260002) and pRhaFlp (BBa_K260003), FLP expression and thus RFP fluorescence can be controlled by small molecules (anhydrotetracycline and L-rhamnose respectively). It is thus a PoPS measurement device, where tet- and rhamnose- promoters can be compared and FLP activity be titrated.
Notes
A strong promoter was necessary and we use BBa_J23100. An appropriate reading frame for the F3 sites had to be found to prevent premature Stop-codons and hydrophobic amino acid residues. Further, the genes encoding ZeoR and mRFP1 were codon optimised for expression in both E. coli and H. sapiens. For potential use in H. sapiens, the CDS were also made free of polyA sites, exon junctions and CpG dinucleotides where possible. The leading alanine of ZeoR was omitted to have exactly 500 bp between both F3 sites.Source
This part was synthesised by "Mr Gene" (Geneart) and is available in the following plasmid backbones:pMA-RQ-@TetFlp (BBa_K260009)
pTetFlp (BBa_K260002)
pRhaFlp (BBa_K260003)
| Sequence Annotation | Location | Component / Role(s) |
| BBa_J23100 (strong promoter) BBa_B0034 (Elowitz RBS) ATG-Start translated F3 site optimised ZeoR BBa_B0015 (double terminator) F3 site optimised mRFP1 (without Start-codon) | 1,35 44,55 62,64 66,99 101,469 470,598 600,633 635,1309 | promoter feature/promoter feature/rbs ribosome_entry_site start_codon feature/start feature/misc sequence_feature feature/cds CDS feature/stem_loop stem_loop sequence_feature feature/misc CDS feature/cds |