Part:BBa_K404120

[AAV2]-left-ITR_pCMV_betaglobin_CFP_hGH_[AAV2]-right-ITR

[AAV2-left-ITR_pCMV_betaglobin_CFP_hGH_[AAV2]-right-ITR ]
BioBrick Nr.	BBa_K404120
RFC standard	RFC 10
Requirement	pSB1C3
Source	pAAV_MCS provided by Stratagene
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

Producing recombinant virus particles for therapeutical applications is, besides specific cell targeting, purification and quantification assays of AAV-2, one intention of the Virus Construction Kit provided by the iGEM team Freiburg_Bioware 2010. For obtaining a modular toolkit, the complex biological system of the Adeno-associated virus serotype 2 was examined by an exhaustive literature search. Subsequently, the essential components for AAV-2 particle production were extracted and redesigned to match the iGEM standard.

The provided tripartite system is independent of a superinfection  of Adeno- or herpes simplex viruses since the genes encoding the required helper-proteins are co-transfected. Inside the eukaryotic host cell, the DNA sequence containing the inverted terminal repeats (ITRs) is extracted and later encapsidated into the preformed capsids after production of single-stranded DNA. Consequently, this plasmid is known as the vector plasmid (pGOI). Promoter, beta-globin intron and the hGH terminator signal are flanked by the ITRs (ITRs, BBa_K404100 and BBa_K404101) and regulate transgene expression. The vector plasmid containing the desired gene of interest is cotransfected with the RepCap plasmid (BBa_K404001, BBa_K404002 or BBa_K404003) and the pHelper plasmid. To obtain the fully assembled vector plasmid, several assembly steps have to be performed. 

The Composite part [AAV2]-left-ITR_pCMV_betaglobin_CFP_hGH_[AAV2]-right-ITR provided by the iGEM team Freiburg_Bioware comprises all cis-elements required for efficient transgene expression in mammalian cells (polyadenylation terminator: BBa_K404108 and transcription enhancer element: BBa_K404107) and encapsidation into virus particles (ITRs: BBa_K404100 and BBa_K404101). The CFP coding sequence as gene of interest enables facile detection of transduced cells using flow cytometry or fluorescence microscopy.

 

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1319
  Illegal AgeI site found at 2039
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2441