Difference between revisions of "Part:BBa K404255"

Revision as of 00:37, 28 October 2010

pCMV_[AAV2]-VP123ex (ViralBrick-587KO-Z34C)
The Z34C antibody binding motif including a knockout of the natural HSPG tropism BBa_K404213, inserted into the 587 loop of pCMV_[AAV2]-VP123

Usage and Biology

ViralBrick-587KO-Z34C

The Z34C antibody binding motif is a 34 amino acids long fragment from the a minimized fragment of the Z Domain of Staphylococcal Protein A. It specifically binds to the Fc-Domain of antibodies. If inserted into AAV capsids, it can bind antibodies against a specific antigen, allowing differential targeting without the need for further genetic engineering.

[AAV2]-VP123ex

The pSB1C3_001_CMV_VP123 contains a CMV promoter upstream the VP123 sequence. The AAV capsid consists of 60 capsid protein subunits. The three cap proteins VP1, VP2, and VP3 are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Alternative splicing and translation initiation of VP2 at a nonconventional ACG initiation codon promote the expression of VP1, VP2 and VP3. The VP proteins share a common C terminus and stop codon, but begin with a different start codon. The N termini of VP1 and VP2 play important roles in infection and contain motifs that are highly homologous to the phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).
CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues [Fitzsimons et al., 2002].

References

DiPrimio, Asokan, Govindasamy, Agbandje-McKenna, & Samulski, June 2008. Surface loop dynamics in adeno-associated virus capsid assembly. Journal of virology, 167(1), 5178–5189
Fitzsimons, H.L., Bland, R.J. & During, M.J. 2002. Promoters and regulatory elements that improve adeno-associated virus transgene expression in the brain. Methods San Diego Calif, 28(2), pp.227-236. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12413421.

Figure 1: The VP proteins are encoded in an overlapping open reading frame.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 2396
Illegal XhoI site found at 698
Illegal XhoI site found at 884
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 665
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 3024
Illegal SapI site found at 1833

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K404255 short</partinfo>
-<br><b>The Z34C antibody binding motif including a knockout of the natural HSPG tropism, inserted into the 587 loop of [[Part:BBa_K404007|pCMV_[AAV2]-VP123]]</b>
+<br><b>The Z34C antibody binding motif including a knockout of the natural HSPG tropism [https://parts.igem.org/Part:BBa_K404213 BBa_K404213], inserted into the 587 loop of [[Part:BBa_K404007|pCMV_[AAV2]-VP123]]</b>
@@ Line 8: / Line 8: @@
 ===Usage and Biology===
 <html>
+<h3>ViralBrick-587KO-Z34C</h3><br/>
 <div style="width:965px; height:200px; ">
 <div style="float:left; width:460px; height:auto;  margin: 0px 5px 0px 5px; text-align:justify;">
@@ Line 14: / Line 15: @@
 </div>
 <div style="float:right; width:480px; height:auto; "><img src="https://static.igem.org/mediawiki/parts/9/98/Freiburg10_ViralBrick_motif_Z34C.png" width="460"
-height="auto"/></div></div>
+height="auto"/></div></div><br/><br/>
+<h3>[AAV2]-VP123ex</h3> <br/>
+The pSB1C3_001_CMV_VP123 contains a CMV promoter upstream the VP123 sequence. The AAV capsid consists of 60 capsid protein subunits. The three cap proteins VP1, VP2, and VP3 are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Alternative splicing and translation initiation of VP2 at a nonconventional ACG initiation codon promote the expression of VP1, VP2 and VP3. The VP proteins share a common C terminus and stop codon, but begin with a different start codon. The N termini of VP1 and VP2 play important roles in infection and contain motifs that are highly homologous to the phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).
+<br/>
+CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues [Fitzsimons et al., 2002].
+<h3>References</h3>
+<b>DiPrimio, Asokan, Govindasamy, Agbandje-McKenna, & Samulski</b>, June 2008. Surface loop dynamics in adeno-associated virus capsid assembly. Journal of virology, 167(1), 5178–5189 <br />
+<b>Fitzsimons, H.L., Bland, R.J. & During, M.J.</b> 2002. Promoters and regulatory elements that improve adeno-associated virus transgene expression in the brain. Methods San Diego Calif, 28(2), pp.227-236. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12413421. <br />
+<center><img src="https://static.igem.org/mediawiki/parts/a/a7/Freiburg10_Cap_proteins_VP1_2%263.png" width="600"
+height="auto"/></center>
+<b> Figure 1: The VP proteins are encoded in an overlapping open reading frame. </b>
 </html>