Part:BBa_K4421030
pSV40-UAS-iCASP9-2A-GFP
An inducible caspase-9 suicide gene was placed under the control of a combined UAS-SV40 promoter. The GFP tag serves as a reporter and the 2A peptide sequence was intercalated between iCASP9 and the GFP tag to allow the fusion peptide chain to separate into two peptide chains.
Special Note
To simplify the transfection process and improve the transfection efficiency, we decided to transfect pSV40-UAS-iCASP9-2A-GFP(BBa_K4421030) and NFAT_RE-Gal4-KRAB(BBa_K4421029) on the same plasmid. But the KRAB protein has been demonstrated to be capable of inhibiting all promoters within at least 3 kB and the forward construct would therefore inhibit the NFAT-RE promoter, we generated the opposite construct, in which the two expression cassettes were cloned in such a manner that both promoters were at opposite ends and at a long distance.
The mechanism and usage of pSV40-UAS-iCASP9-2A-GFP
This part is placed downstream of NFAT_RE-Gal4-KRAB(BBa_K4421029) and the presence of Gal4-KRAB can inhibit the promotor pSV40-UAS(BBa_K4040003), thus diminishing the synthesis of the iCASP9(BBa_K4421002).The engineered cells can survive if the concentration of iCASP9 is low enough after AP1903 addition.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 781
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 463
Illegal BsaI.rc site found at 1603
Illegal BsaI.rc site found at 2514
| None |