Part:BBa_K5471013

Cassette 2 (ITR-EF1a-mir195-eGFP-SV40pA-ITR)

This cassette is an autonomous device for the simoultaneous expression of microRNA 195 and eGFP reporter gene protein (Cassette 1 https://parts.igem.org/Part:BBa_K5471012), and specially placed in between a pair of ITR sequences for the AAV2 serotype(https://parts.igem.org/Part:BBa_K5471004).

This cassette is comprised of (from 5' end); 5' end ITR, an EF1a mir195 infused promoter(https://parts.igem.org/Part:BBa_K5471011 , https://parts.igem.org/Part:BBa_K5471009 , https://parts.igem.org/Part:BBa_K5471005), a kozak sequence(https://parts.igem.org/Part:BBa_K5471006) followed by the coding region of eGFP(https://parts.igem.org/Part:BBa_K5471007), and SV40 polyadenylation signalling sequence(https://parts.igem.org/Part:BBa_K5471008) for the termination of transcription, 3' end ITR.

The EF1a mir195 infused promoter is responsible for driving the intragenic expression mir195 inside the EF1a intron and at the same time is responsible for the strong transcription of the eGFP protein gene. This cassette provides the ability to efficiently express a micro-RNA and at the same time express a reporter protein that confirms the expression of the micro-RNA.

However, both the micro-RNA and reporter gene can be swapped with other Parts in the future by anyone wishing to improve on this part.

The ITR pair is flanking makes the cassette ideal for rAAV serotype 2 production, when it is used for the construction of an appropriate Transfer-Gene plasmid. The ITR pair promises the full insertion of the cassette they flank to be inside the viral DNA of the rAAV2 particles that are produced.

This composite part's sequence was used for the construction of the pAAV-EF1a-mir195-eGFP-SV40pA plasmid https://parts.igem.org/Part:BBa_K5471020 our team used for our experiments

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 587
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2136
  Illegal PstI site found at 587
  Illegal NotI site found at 2130
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 735
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 587
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 587
  Illegal NgoMIV site found at 470
  Illegal AgeI site found at 195
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1240