Part:BBa_K300002:Design
Phasin (PhaP) - head domain
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
It is identical to BBa_K208001, but it lacks the stop codon in order to support protein fusions.
It has been designed as a head domain:
The Prefix sequence is 5'-GAATTCGCGGCCGCTTCTAG-3' (RFC10 Prefix)
The Suffix sequence is 5'-ACTAGTAGCGGCCGCTGCAG-3' (RFC23 Suffix)
For these reasons, a tail domain or an internal domain (compatible with RFC23) can be easily assembled downstream to create protein fusions.
CONSTRUCTION METHOD:
BBa_K208001 (provided by iGEM HQ in pSB3K3 in RFC23 standard) was PCR-amplified/mutagenized with primers:
phaP10F 5'-GCTTCTAGATGATCCTCACCCCGGAACA-3'
phaPSR: 5'-GCTACTAGTGGCAGCCGTCGTCTTCTTTG-3'
in order to delete the stop codon.
The PCR product was ran on a 1% agarose gel, gel-extracted, cut with XbaI-SpeI and ligated with pSB1AK3 (previously cut with XbaI-SpeI and dephosphorylated). Positive clones were found through digestion screening/sequencing.
Source
Ralstonia eutropha genomic DNA and BBa_K208001.
References
Banki MR, Gerngross TU, Wood DW (2005), Novel and economical purification of recombinant proteins: Intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association. Protein Sci. June; 14(6): 1387–1395. doi: 10.1110/ps.041296305.