Part:BBa_K415514

pCMV L4R1 MammoBlock Entry Vector

MammoBlock L4R1 Promoter vector. Contains Human cytomegalovirus (CMV) promoter, one of the strongest promoters described. Based on RNA polymerase II system, CMV promoter drives higher-level constitutive expression of genes in a broader variety of mammalian cell lines, compared with RNA polymerase III based promoters such as U6 and H1.

Characterization

Figure 1. Production of EGFP in HEK293FT cells post calcium phosphate transfection with CMV_EGFP.

Calcium Phosphate Transfection of HEK293FT Cells with CMV_EGFP Constructs.

DNA, CaCl2, water, and HBSS were mixed to form calcium-phosphate bound DNA particles for uptake. Presence of precipitate was confirmed under brightfield micrsocope. Cells were incubated overnight in cell incubator at 37C, 5% CO2. Micrographs with brightfield and fluorescence were recorded roughly 24 hours post transfection. Exposure times: brightfield 5.2ms, GFP 600 ms.

Results

CMV exhibits a striking amount of transcriptional activity shortly after calcium phosphate transfection, as seen in Figure 1. The activity increases drastically within 48 hours. Further observation has shown that activity remains high constitutively with time.

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           

Figure 2. Production of EGFP in HEK293FT cells post infection with CMV_EGFP.

Infection of HEK293FT cells with CMV_EGFP constructs.

Suspension of 20,000 cells/well in 0.5 mL DMEM + surfectant in 24-well plate were infected with replication-deficient HIV virus particles carrying the CMV_EGFP construct. Medium change occured immediately after overnight incubation, and micrographs were taken roughly 24 and 48 hours after infection on Zeiss fluorescence microscope. Exposure times: brightfield 5.2 ms, GFP 199 ms.

Results

CMV exhibits a striking amount of transcriptional activity shortly after viral infection, as seen in Figure 2. The activity increases drastically within 48 hours. Further observation has shown that activity remains high constitutively with time.                                                                                                                                                                                                                                                                                                                                                                                              

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 601
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 601
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 601
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 601
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 601
  
• 1000
  COMPATIBLE WITH RFC[1000]