sfGFP.degradation.tag under Pta promoter in psb1C3_IntKR plasmid
AANDENYALAA. (Very fast) SsrA degradation tag.
C-terminal degradation tags are commonly found in high turnover proteins in Escherichia coli.
This sequence codes for the amino acid sequence AANDENYALAA, which when fused to the C-terminal of proteins, will make the protein susceptible to very fast degradation through SspB-mediated binding to the ClpX protease. The following rates of degradation of this tag are pulled from the corresponding references below: ~5 Vmax/ [Clpx6] min-1 from (1) ~0.5%/min on log scale from (2) ~1 min half life from (3) See the following references for further information on degradation rates and mechanisms of this tag: (1)McGinness, Baker, Sauer. 2006. Mol. Cell. 22:701. (2)Flynn et al 2003. Mol. Cell. 11: 671. Flynn et al. 2001. PNAS 98(19): 10584. Anderson et al 1998. App. Env. Microbiol. 64(6):2240.
C-terminal tag. Degradation rate is very fast. Deviations from this sequence in key amino acids will lower degradation rates (see Parts BBa_M0051, BBa_M0052, BBa_M0053). Three C-terminal aa's (LAA in this case) are necessary and sufficient for ClpX binding and degradation. Upstream aa sequence serves as a binding site for SspB, which guides rapid binding to ClpX.
Superfolder GFP was originally described by: Pedelacq et al (2006): "Engineering and characterization of a superfolder green fluorescent protein", Nature Biotech 24 (1) January 2006 This version was synthesised de novo (by Geneart).
This is the coding sequence of superfolder GFP (Pedelacq et al (2006): "Engineering and characterization of a superfolder green fluorescent protein", Nature Biotech 24 (1) January 2006). It carries the following amino acid changes with respect to mut3 GFP (E0040), the currently most commonly used GFP in the registry: S30R, Y39N, F64L, G65T, F99S, N105T, Y145F, M153T, V163A, I171V, A206V Its in-vivo properties are considerably improved with respect to mut3 - it develops fluorescence about 3fold faster than mut3 GFP and reaches 4fold higher absolute fluorescence levels. Fluorescenct colonies can be identified with the naked eye even without UV or blue light illumination (that is to say the amount of blue light in normal daylight or lablight is sufficient). Additionally it is more stable in vitro and refolds faster after in vitro denaturation with respect to mut3 GFP. Note: Superfolder GFP is available in constructs driven by the pBAD and T7 promoters: part numbers I746908 and I746909 respectively. Additionally 6-his tagged versions for protein purification exist: I746914 (pBAD driven) and I746915 (T7 driven).
Codon optimisation before de novo synthesis was carried out for both, E.coli and Bacillus subtilis.
Synechocystis PCC6803 neutral recombination site.
PCR amplified from synechocystis PCC6803 chromosome.
Any DNA sequence flacked by this part and BBa_K743001 will be incorporated into Synechocistys PCC6803 chromosome through a neutral double homologous recombination.
According to literature, in Synechocystis the recombination sites must be at least 450bp long to allow a rasonably high transformation efficiency, being 700bp the optimum size.
PCR amplified from Synechocystis PCC6803 chromosome, orf slr0337
When placed between this part and BBa_K743000, any DNA sequence will be inserted into Synechocystis PCC6803 chromosome trough homologous recombination
According to literature, in Synechocystis the recombination sites must be at least 450 bp long to allow a rasonably high transformation efficiency, being approximately 1200 bp the optimum size.
Pta promoter and native rbs from Synechocystis PCC6803
This part was PCR amplified from Synechocystis chromosome, orf slr1739.
This is the 150bp sequence upstream the Transaldolase gene start codon, it contains putative rbs and promoter regions. Transaldolase is an enzyme involved in the pentose phospate cycle and its mRNA levels have shown to oscillate in a circadian manner, peaking at hour 14.
It was assumed that the 150bp sequence upstream from the start codon includes the promoter and the ribosome binding site
Double terminator consisting of BBa_B0010 and BBa_B0012
This contains KanR, a terminator and then AmpR and a terminator.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1