Part:BBa_K3200999

hCMV promoter and IgK leader sequence and PDGFR transmembrane domain for membrane protein

This vector is based on mammalian pDisplay vector of invitrogen, and has been modified to comply with RFC10. This vector includes prefix/surfix, pUC origin, Neomycine/Kanamaycin resistance gene, PDGFR transmembrane domain, pcDNA3.1/BGH reverse priming site, BHG polyA addition site, hCMV promoter/enhancer region, IgK leader sequence, T7 promoter priming site, TATA, CAAT and AP1 sequences.

Prefix and suffix contains several restriction enzyme sites that replace the MCS to meet RFC10. An rhodopsin-DRD2 chimeric gene fragment is inserted into this region. 
For pUC origin, this is a typical origin of replication sequence for cloning vectors, derived from the University of Calfinornia. The ori sequence itself was made by inducing a single point mutation in the ori of plasmid pMB1 and has a very high copy number. Neomycine / Kanamycin is a sequence for selecting transformed and transfected cells when cultured in a medium or preventing contamination by foreign bacteria.
The Ig-κ leader sequence is a signal sequence that functions at the N terminus of a protein and initiates a translocation signal to the endoplasmic reticulum membrane so that the protein can be secreted or embedded in the membrane (Coloma et al., 1992). Therefore, it should be located upstream of the gene fragment and prefix.
The platelet-derived growth factor receptors (PDGFR) transmembrane domain is a hydrophobic transmembrane region of cell surface tyrosine kinase receptors and functions as a membrane anchor or signal-anchor sequence. Proteins secreted or inserted into plasma membranes, vesicle membranes, etc. should be expressed through co-translational translocation pathways. Therefore, when translated in ribosomes, signal recognition protein (SRP) should be preferentially translated at the N terminus. SRP then translocates the protein into the endoplasmic reticulum. The membrane anchor sequence is a stop-transfer sequence that stops translocation when the protein is inserted into the membrane to some extent. Without this sequence, the protein's membrane penetration would not stop, and thus would not be expressed on the plasma membrane but secreted out of the cell. This stop function of the anchor protein is located downstream of the IgK leader sequence and prefix / suffix because it must function after membrane insertion.
TATA box and CAAT box are consensus sequences that designate transcription start sites in eukaryotes, respectively. They are highly conserved in eukaryotes. TATA boxes are essential for expression and, for CAAT, are necessary for sufficient expression. It interacts with hCMV, a promoter and enhancer of eukaryotes, to promote gene fragment expression. Transcription factors bind to the AP1 sequence to specifically promote transcription.
 This vector also has two priming site, the T7 promoter priming site and the pcDNA3.1 / BGH reverse priming site. 

Experiments

 In the experiments, we treated the DiOC2 reagent to observe the change in membrane potential of U-87 MG cells. DiOC2 becomes red when the membrane potential is hyperpolarized in the plasma membrane of the cell and blue when depolarized. Three cells were cultured in 90% EMEM, 9% FBS, 1% PENstrep. Before the experiment, we tested the DiOC2 by CCCP, which destroy the membrane potential and make the fluorescence image all red.
  The transfected and WT cells were incubated at 1 h and 37 ° C. under light and dark conditions, respectively, and then treated with DiOC and incubated at 37 ° C. for 15 minutes. Light conditions were given only 4x4 LEDs (blue light) in a dark box.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5240
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 5246
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5240
  Illegal BamHI site found at 5122
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 5240
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 5240
  Plasmid lacks a suffix.
  Illegal XbaI site found at 5255
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 1626
  Illegal NgoMIV site found at 1909
  Illegal NgoMIV site found at 4108
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 1414
  Illegal BsaI.rc site found at 3131
  Illegal BsaI.rc site found at 5097
  Illegal SapI site found at 1849