Project

Part:BBa_K404118

Designed by: Freiburg Bioware 2010   Group: iGEM10_Freiburg_Bioware   (2010-10-11)
Revision as of 15:37, 27 October 2010 by Jessica (Talk | contribs)

[AAV2]-left-ITR_phTERT_betaglobin

[AAV2-left-ITR_pCMV_betaglobin]
Freiburg10 Vectorplasmid precursors 5.png
BioBrick Nr. BBa_K404118
RFC standard RFC 10
Requirement pSB1C3
Source pAAV_MCS provided by Stratagene
Submitted by [http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]


Freiburg10 Vectorplasmid precursors 5.png














[AAV2]-left-ITR_phTERT_betaglobin

 

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. 

Several assembly steps have to be performed in order to obtain the fully assembled vector plasmid ensuring a layer of specificity by using a tumor-specific promoter phTERT (BBa_K404106).  Facilitating the cloning steps the iGEM team Freiburg_Bioware 2010 provide the 5´nucleotide components in respect to the desired gene of interest which are the left inverted terminal repeat (BBa_K404100) followed by the phTERT promoter (BBa_K404106) and a putative enhancer-element (BBa_K404107).

 







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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//chassis/eukaryote/human
//viral_vectors/aav
//viral_vectors/aav/vector_plasmid
Parameters
None