|5' 测序引物及序列:||T7 Forward: 5’-TAATACGACTCACTATAGGG-3’|
|3' 测序引物及序列:||TK polyA Reverse: 5’-CTTCCGTGTTTCAGTTAGC-3’|
pLVX-DD-AmCyan1 Control 载体是阳性对照载体，需要与pLVX-DD-AmCyan1 Reporter载体共同使用；
DD-AmCyan1与AmCyan1相比，N端融合了ProteoTuner destabilization domain (DD)去稳定结构域，
pLVX-DD-AmCyan1 Control 载体描述 pLVX-DD-AmCyan1 Control constitutively expresses the destabilized cyan fluorescent protein DD-AmCyan1. The vector can be used as a control to monitor ligand-dependent stabilization of DD-AmCyan1 in your cell-type of interest. The Lenti-X DD-AmCyan1 Vector Set (available as part of the Lenti-X DD Cyan Reporter System; Cat. No. 631748) includes two HIV-1-based, lentiviral expression vectors that can efficiently transduce both dividing and nondividing mammalian cells. This reporter set can be used to monitor promoter activity in live cells and in vivo. pLVX-DD-AmCyan1 Reporter is a promoterless vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS). The gene downstream of the MCS encodes the cyan fluorescent protein DD-AmCyan1, a modified version of AmCyan1 that is tagged on its N-terminus with the ProteoTuner? destabilization domain (DD; 1). In the absence of the Shield1 ligand, the DD tag induces rapid degradation of the fluorescent reporter, minimizing any background caused by leaky promoters; but upon addition of Shield1 at the time of promoter activation, the DD-tagged reporter molecules are stabilized, increasing the signal-to-noise ratio. pLVX-DD-AmCyan1 Control drives reporter expression via a constitutive promoter, and thereby serves as a positive control. DD-AmCyan1 Reporter AmCyan1 (excitation and emission maxima: 453 and 486, respectively) is a human codon-optimized variant of the wildtype Anemonia majano cyan fluorescent protein (AmCyan) that exhibits enhanced emission characteristics (2, 3). DDAmCyan1 is tagged on its N-terminus with the ProteoTuner DD, which causes rapid, proteasomal degradation of DDAmCyan1. However, when the membrane-permeant, stabilizing ligand Shield1 is added to the medium, it binds to the DD and prevents degradation of the DD-AmCyan1 reporter protein, thereby causing it to accumulate inside the cell. In the absence of the stabilizing ligand Shield1, the DD causes the degradation of any DD-AmCyan1 reporter protein produced prior to promoter activation, thus minimizing background fluorescence caused by leaky promoters. To analyze promoter activity, the inducer of choice is added to the medium along with Shield1, which effectively stabilizes the reporter protein, allowing it to accumulate. As a result, only the reporter molecules expressed during promoter induction will contribute to the fluorescence signal, providing a considerably higher signal-to-noise ratio than that obtained with non-destabilized or constitutively destabilized reporter systems. For both vectors, the promoter’s activity level can be directly correlated to the fluorescence level. Lentiviral Elements The reporter and control vectors each contain all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral and transgene RNA (4), leading to increased viral titers from packaging cells, and enhanced expression of your gene of interest in target cells. In addition, each vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RNA out of the nucleus (5). Finally, each vector also contains a central polypurine tract/central termination sequence element (cPPT/CTS). During target cell infection, this element creates a central DNA flap that increases nuclear import of the viral genome, resulting in improved vector integration and more efficient transduction (6). Lentiviral particles derived from the vectors allow you to monitor your promoter of interest in virtually any cell type, even primary cells. Antibiotic Selection In addition to lentiviral elements, the reporter and control vectors each contain a puromycin resistance gene (Puror) under the control of the murine phosphoglycerate kinase promoter (PPGK) for the selection of stable transductants. The vectors also contain pUC origins of replication and E. coli ampicillin resistance genes (Ampr) for propagation and selection in bacteria. Propagation in E. coli Recommended host strains: DH5α and other general purpose strains. Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts. E. coli replication origin: pUC Copy number: high Excitation and Emission Maxima of AmCyan1 Excitation: 453 nm Emission: 486 nm References 1. Banaszynski, L. et al. (2006) Cell 126(5):995–1004. 2. Matz, M. V. et al. (1999) Nature Biotech. 17(10):969–973. 3. Haas, J. et al. (1996) Curr. Biol. 6(3):315-324. 4. Zufferey, R. et al. (1999) J. Virol. 73(4):2886–2892 5. Cochrane, A. W. et al. (1990) Proc. Natl. Acad. Sci. USA 87(3):1198–1202. 6. Zennou, V. et al. (2000) Cell 101(2):173–185.