Part:BBa_K1470006

pMIG

Template pMIG

Usage and Biology

pMIG is a novel and easy way to handle mammalian expression vectors and to create fast and efficiently stable cell lines. It contains Long Terminal Repeats (LTR) for the production of viral vectors. There is no packaging signal or gag, pol, env in pMIG. Thus it can be used in any biosafety level 1 laboratory.

Origin, Standardization and Application

This plasmid was originally created by William Hahn and is available as plasmid 9044 at Addgene. It is a high copy plasmid and contains an Ampicillin resistance.

In order to provide this plasmid as a mammalian expression vector, we removed several EcoRI- and PstI-sites and an IRES. Afterwards we inserted a multiple cloning site, consisting of XhoI, EcoRI, NotI, SalI, PstI, HindHIII and ClaI. However, it was not possible to delete Xba I-sites in 3'- and 5' LTRs due to their nearly identical sequences. Nonetheless, any biobrick cut with EcoRI/PstI can be ligated in pMIG, hence providing future iGEM Teams a well functioning and reliable expression vector for mammalian cells.

Because of pMIG's retroviral origins, it can be used only in packaging cell lines such as Phoenix cells to produce viral vectors. Otherwise there will not be any functional vectors due to the lack of gag, pol and env.No additional promotor is required for expression on the account of 3'LTR acting as one.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4917
  Illegal XbaI site found at 289
  Illegal XbaI site found at 3711
  Illegal PstI site found at 10
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4917
  Illegal NheI site found at 99
  Illegal NheI site found at 3521
  Illegal PstI site found at 10
  Illegal NotI site found at 4925
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4917
  Illegal BglII site found at 4899
  Illegal XhoI site found at 4905
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4917
  Illegal XbaI site found at 289
  Illegal XbaI site found at 3711
  Illegal PstI site found at 10
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4917
  Illegal XbaI site found at 289
  Illegal XbaI site found at 3711
  Illegal PstI site found at 10
  Illegal NgoMIV site found at 3466
  Illegal NgoMIV site found at 4123
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 520
  Illegal BsaI site found at 541
  Illegal BsaI site found at 3964
  Illegal BsaI.rc site found at 1901
  Illegal BsaI.rc site found at 4030
  Illegal BsaI.rc site found at 4645
  Illegal SapI site found at 818