Part:BBa_K1460001:Design
pT7 + RBS + GST (glutathione-S-transferase)-CRS5 (metallothionein) + Ter
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 764
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 176
Design Notes
Flanking the T7 promoter are two KpnI cut sites with both the T7 promoter and a SacI cut site between them. These cut sites are present for easy removal of the T7 promoter for modulation of parts with other promoters. The SacI cut site can be used as a marker for successful removal of the T7 promoter from the part. When digested with SacI, part BBa_K1460001 in pSB1C3 will yield bands of size 2166bp and 931bp. If the T7 promoter is successfully removed, when digested with SacI the plasmid will yield the linearized length of the pSB1C3+BBa_K1460001
Source
T7 promoter is part BBa_I712074. RBS is from part BBa_J61101. Glutathione-S-transferase is from S. japonicum. CRS5 is a metallothionein from S. cerevisiae. Terminator is part BBa_B1006. This sequence was synthesized by GenScript in the vector pUC57 and was then transferred using traditional cloning into pSB1C3.
References
[1] Smith, D., Davern, K., Board, P., Tiu, W., Garcia, E., & Mitchell, G. (1986). Mr 26,000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase. Proc. Natl. Acad. Sci. U.S.A., 83(22), 8703-8707.
[2] Culotta, V., Howard, W., & Liu, X. (1994). CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae. J. Biol. Chem., 269(41), 25295-25302.
[3] Johnsonb, K. S. (1988). Single-step purification S-transferase, 67, 31–40.