Part:BBa_K4006013
MT-6XHis-IEE5-arsC-HA-IEE2-FLAG-fTNa
Background
This composite part includes coding sequences for the proteins metallothionein, arsenate reductase, and the protein ferritin, all codon optimized for use in the Chlamydomonas reinhardtii chloroplast. They were also each tagged with a different epitope tag, for the eventual purpose of protein expression evaluation by Western blot. The intention was for them to work synergistically to improve the uptake of arsenic out of contaminated water.
Cloning into E. coli and Verification
The constructs were designed by our team and produced by IDT. They were transformed into 10-Beta competent E. coli cells from NEB. The “Tagged” sequence, MT/6xHIS-IEE2-ArsC/HA-IEE5-FtnA/FLAG, was designed by our team and produced in two parts by IDT. However, our team did not have any success cloning this construct via golden braid. This may be due to the presence of IEE2, which is also present in the pASapI backbone. We were unable to confirm cloning via restriction digest and often did not get significant results from the transformation into 10-beta competent E. coli cells. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 589
Illegal BamHI site found at 45 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 5
Illegal SapI site found at 685
Illegal SapI site found at 1750
Illegal SapI.rc site found at 233
Illegal SapI.rc site found at 1150
Illegal SapI.rc site found at 2305
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