Difference between revisions of "Part:BBa K404013"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K404013 short</partinfo>
 
<partinfo>BBa_K404013 short</partinfo>
  
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{| style="color:black; margin: 0px 0px 500px 20px;" cellpadding="6" cellspacing="1" border="2" align="right"
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! colspan="2" style="background:#66bbff;"|[https://parts.igem.org/Part:BBa_K404013 pCMV_AAV2-VP3]
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|-
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|'''BioBrick Nr.'''
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|[https://parts.igem.org/Part:BBa_K404013 BBa_K404013]
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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_001<br>
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|-
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|'''Source'''
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|pAAV_RC from 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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<html>
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The viral capsid is formed by the three structural proteins VP1, VP2 and VP3 which are encoded by the cap gene in an overlapping reading frame. They form an icosahedral symmetry arranged in a stoichiometric ratio of 1:1:10. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Sharing a common C terminus and stop codon, the VP proteins begin with a different start codon. The translation of VP2 from the major spliced mRNA is not as efficient as of VP1 and VP3 because it initiates at a Thr codon (ACG). The N terminus of VP2 contains a motiv of about 70 amino acids that is highly homologous to a phospholipase A2 (PLA2) domain.  Furthermore, there are nuclear localization sequences (BR)(+) which are supposed to be necessary for endosomal escape and nuclear entry. The exact role of VP2 remains unknown, although the protein is thought to be nonessential for viral assembly and infectivity.
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<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 />
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<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" margin: 10px 10px 10px 10px/></center>
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<b> Figure 1: The VP proteins are encoded in an overlapping open reading frame. </b>.
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===Usage and Biology===
 
  
 
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Latest revision as of 16:50, 30 October 2010

pCMV_[AAV2]-VP3


pCMV_AAV2-VP3
BioBrick Nr. BBa_K404013
RFC standard RFC 10
Requirement pSB1C3_001
Source pAAV_RC from Stratagene
Submitted by [http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

The viral capsid is formed by the three structural proteins VP1, VP2 and VP3 which are encoded by the cap gene in an overlapping reading frame. They form an icosahedral symmetry arranged in a stoichiometric ratio of 1:1:10. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Sharing a common C terminus and stop codon, the VP proteins begin with a different start codon. The translation of VP2 from the major spliced mRNA is not as efficient as of VP1 and VP3 because it initiates at a Thr codon (ACG). The N terminus of VP2 contains a motiv of about 70 amino acids that is highly homologous to a phospholipase A2 (PLA2) domain. Furthermore, there are nuclear localization sequences (BR)(+) which are supposed to be necessary for endosomal escape and nuclear entry. The exact role of VP2 remains unknown, although the protein is thought to be nonessential for viral assembly and infectivity.
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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 665
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1230