Part:BBa_K404009

pCMV_[AAV2]-VP1

pCMV_AAV2-VP1
BioBrick Nr.	BBa_K404009
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. Whereas VP1 is translated from the minor spliced mRNA, VP2 and VP3 are translated from the major spliced mRNA. The minor spliced product is approximately 10-fold less abundant than the major spliced mRNA. Thus, there is much less VP1 than VP2 and VP3 resulting in a capsid stoichiometric ratio of 1:1:10. The N terminus of VP1 has an extension of 65 amino acids including an additional extension of 138 N-terminal amino acids forming the unique portion of VP1. It contains a motiv of about 70 amino acids that is highly homologous to a phospholipase A2 (PLA2) domain. Furthermore, there are nuclear localization sequences (NLS) which are supposed to be necessary for endosomal escape and nuclear entry.
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

Bleker, Pawlita, & Kleinschmidt, 2006. Impact of capsid conformation and rep-capsid interactions on adeno-associated virus type 2 genome packaging. Journal of Virology, 810–820
b>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
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 XhoI site found at 701
  Illegal XhoI site found at 887
  
• 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 1836