Difference between revisions of "Part:BBa K404255"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K404255 short</partinfo>
 
<partinfo>BBa_K404255 short</partinfo>
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<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>
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<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></b>
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{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="left"
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! colspan="2" style="background:#66bbff;"|[https://parts.igem.org/Part:BBa_K404255 [AAV2]-left-ITR_pCMV]
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|-
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|'''BioBrick Nr.'''
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|[https://parts.igem.org/Part:BBa_K404255 BBa_K404255]
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|-
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|'''RFC standard'''
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|[https://parts.igem.org/Help:Assembly_standard_10 RFC 10]
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|-
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|'''Requirement'''
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|pSB1C3<br>
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|-
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|'''Source'''
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|pAAV_MCS provided by Stratagene
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|-
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|'''Submitted by'''
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|[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]
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|}
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<br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
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===Usage and Biology===
 
===Usage and Biology===
 
<html>
 
<html>
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<div style="width:965px; height:400px; ">
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<h5>ViralBrick-587KO-Z34C</h5><br/>
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<div style="width:965px; height:200px; ">
 
<div style="float:left; width:460px; height:auto;  margin: 0px 5px 0px 5px; text-align:justify;">
 
<div style="float:left; width:460px; height:auto;  margin: 0px 5px 0px 5px; text-align:justify;">
 
The Z34C antibody binding motif is a 34 amino acids long fragment from the a minimized fragment of the Z Domain of  <a href=http://www.ncbi.nlm.nih.gov/protein/153106>Staphylococcal Protein A</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.
 
The Z34C antibody binding motif is a 34 amino acids long fragment from the a minimized fragment of the Z Domain of  <a href=http://www.ncbi.nlm.nih.gov/protein/153106>Staphylococcal Protein A</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.
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</div>
 
</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"  
 
<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"  
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height="auto"/></div></div>
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height="auto"/></div></div><br/><br/>
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<h5>[AAV2]-VP123ex</h5> <br/>
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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/>
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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].
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<h3>References</h3>
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<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 />
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<center><img src="https://static.igem.org/mediawiki/parts/a/a7/Freiburg10_Cap_proteins_VP1_2%263.png" width="600"
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height="auto"/></center>
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<b> Figure 1: The VP proteins are encoded in an overlapping open reading frame. </b>
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</html>
 
</html>
  

Latest revision as of 16:36, 13 January 2011

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</b>

[AAV2-left-ITR_pCMV]
BioBrick Nr. BBa_K404255
RFC standard RFC 10
Requirement pSB1C3
Source pAAV_MCS provided by Stratagene
Submitted by [http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]













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