Part:BBa_K1722009
shTERT+GFP Composite
hTERT, which is short for human telomerase reverse transcriptase, is a cancer-cell specific promoter. It can be activated inside cancer cells with no effect on normal cells. By mutating four base pairs of hTERT sequence, we achieved an improved promoter with higher promote efficiency named super hTERT(shTERT). As an important component of human telomerase, hTERT express only in tumor cells and other immortal cells which are telomerase positive. Only in tumor cells that can express TERT can the promoter being activated to realize targeted expression of effector gene.
In this plasmid, we construct hTERT with Green Fluorescent Protein(GFP), which is a widely used reporter gene. When shTERT promoter is activated, GFP is produced and be oxidized to fluoresce. By inserting this plasmid into T24 and 5637, two lines of bladder cancer cells, we are able to acquire GFP. Green fluorescent light is detected using confocal laser scanning microscopy. This indicates that hTERT promoter is able to be activated inside bladder cancer cells.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 473
Design Notes
The shTERT promoter that is achieved from Shenzhen Sencond People's Hospital is constructed in psi-Check2 vector. To assemble it with GFP and pSB1C3, we have to design primers with certain restriction enzyme cutting site and amplify it. Then assemble the three parts using 3A Assembly Method.
Source
This composite part is assembled using shTERT and GFP. shTERT promoter is achieved from Shenzhen Second People's Hospital and GFP is achieved from 2015 Distribution Kit. We constructed the two DNA sequence in pSB1C3, the required vector.
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