Part:BBa_K404250

pCMV_[AAV2]-VP123ex (ViralBrick-587-Beta-lactamase)
ß-Lactamase motif, inserted into the 453 loop of pCMV_[AAV2]-VP123

[pCMV_[AAV2-VP123ex(ViralBrick-587-Beta-lactamase]
BioBrick Nr.	BBa_K404250
RFC standard	RFC 10
Requirement	pSB1C3
Source	pAAV_MCS provided by Stratagene
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

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].

Usage & biology

ß-Lactamase (BLA) is an enzyme produced by some bacteria that cleaves the ß-lactam ring of various antibiotics such as penicillin, rendering them inactive. Because the peptides' N- and C-termini are in close proximity to each other, we created a ViralBrick containing BLA to see if capsid assembly still takes place with such a large insert in place. Assembled capsids can easily be detected by a beta-lactamase assay. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2455
  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 3075
  Illegal BsaI site found at 3711
  Illegal SapI site found at 1833