Part:BBa_K1103004:Design
NFκB-IκB-GFP
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 225
Illegal PstI site found at 408 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 225
Illegal PstI site found at 408 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 75
Illegal XhoI site found at 56
Illegal XhoI site found at 62
Illegal XhoI site found at 240 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 225
Illegal PstI site found at 408 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 225
Illegal PstI site found at 408 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 376
Design Notes
It’s of great importance to remove the terminator of IκB, or the expression may stop after the genes of IκB finished translation.
Source
IκB comes from genomic DNA of the DU-145 cell. GFP comes from the vector pEGFP-N1. We use overlap PCR to fuse them.
References
[1]Carlotti F, Dower S K, Qwarnstrom E E. Dynamic shuttling of nuclear factor κB between the nucleus and cytoplasm as a consequence of inhibitor dissociation[J]. Journal of Biological Chemistry, 2000, 275(52): 41028-41034. [2]Tam W F, Sen R. IκB family members function by different mechanisms[J]. Journal of Biological Chemistry, 2001, 276(11): 7701-7704. [3]Tenjinbaru K, Furuno T, Hirashima N, et al. Nuclear translocation of green fluorescent protein-nuclear factor κB with a distinct lag time in living cells[J]. FEBS letters, 1999, 444(1): 1-4. [4]Nelson D E, Ihekwaba A E C, Elliott M, et al. Oscillations in NF-κB signaling control the dynamics of gene expression[J]. Science, 2004, 306(5696): 704-708. [5]Schmid J A, Birbach A, Hofer-Warbinek R, et al. MECHANISMS OF SIGNAL TRANSDUCTION-Dynamics of NFkB and IkBa studied with green fluorescent protein (GFP) fusion proteins. Investigation of GFP-p65 binding to DNA by fluorescence resonance energy[J]. Journal of Biological Chemistry, 2000, 275(22): 17035-17042.