Project

Part:BBa_K404128

Designed by: Freiburg Bioware 2010   Group: iGEM10_Freiburg_Bioware   (2010-10-11)

[AAV2]-left-ITR_pCMV_mVenus_hGH_[AAV2]-right-ITR

[AAV2-left-ITR_pCMV_mVenus_hGH_[AAV2]-right-ITR]
Freiburg10 Vectorplasmid composite 10.png
BioBrick Nr. BBa_K404128
RFC standard RFC 10
Requirement pSB1C3
Source Assembled vectorplasmid
Submitted by FreiGEM 2010

Providing an element assumed to be an enhancer of transgene expression (Nott et al. 2003), the iGEM team Freiburg tested a beta-globin intron derived from the human beta globin gene which can be fused upstream of the desired gene of interest. The beta-globin intron BioBrick consists of a partial chimeric CMV promoter followed by the intron II of the beta-globin gene. The 3´end of the intron is fused to the first 25 bases of human beta globin gene exon 3. The beta globin intron BioBrick is assumed to enhance eukaryotic gene expression (Nott et al. 2003).

Figure 1: Assembled vectorplasmid lacking the beta-gobin intron.


Characterization

Analysis was conducted as described for the hGH terminator experiment (see above). The vectorplasmid missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral genomes containing the beta-globin intron. Considering these results and taking into account that a constant volume of viral particles has been used for transduction, the difference between the construct containing and lacking the beta-globin intron is minimal. Since packaging efficiency of the AAV-2 decreases with increasing sizes of the insert (Dong et al. 1996), the iGEM team Freiburg_Bioware suggests using the beta-globin intron in dependence on the size of your transgene.

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 822
    Illegal AgeI site found at 1542
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1944


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