Part:BBa_K1722008
hTERT+GFP Composite
Telomere is a special structure in eukaryotic chromosome consisting of (TTAGGG)n and telomere binding protein. Telomerase is a kind of reverse transcript DNA polymerase rely on RNA. It can synthesize repeating nucleotide sequence and add it to the end of the chromosome to maintain the telomere. In this way, cells with telomerase can proliferate limitless and be immortalized. The human telomerase enzyme complex consists of human telomerase reverse transcriptase(hTERT) and human telomerase RNA(hTR). hTR is expressed in almost all cells while hTERT express only in tumor cells and other immortal cells. hTERT promoter, which has tumor specificity, has significantly higher transcription activity in telomerase positive tumor cells. Research shows that targeted gene therapy based on hTERT promoter can selectively kill telomerase positive tumor cells with no influence on normal cells. In this plasmid, we construct hTERT with Green Fluorescent Protein(GFP), which is a widely used reporter gene. When hTERT 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 hTERT 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 hTERT and GFP. hTERT 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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