Difference between revisions of "Part:BBa K404007"
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| + | ! colspan="2" style="background:#66bbff;"|[https://parts.igem.org/Part:BBa_K404008 pCMV_AAV2-VP123] | ||
| + | |- | ||
| + | |'''BioBrick Nr.''' | ||
| + | |[https://parts.igem.org/Part:BBa_K404007 BBa_K404007] | ||
| + | |- | ||
| + | |'''RFC standard''' | ||
| + | |[https://parts.igem.org/Help:Assembly_standard_10 RFC 10] | ||
| + | |- | ||
| + | |'''Requirement''' | ||
| + | |pSB1C3_001<br> | ||
| + | |- | ||
| + | |'''Source''' | ||
| + | |pAAV_RC from Stratagene | ||
| + | |- | ||
| + | |'''Submitted by''' | ||
| + | |[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010] | ||
| + | |} | ||
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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. The VP1 N-terminus contains motifs that are highly homologous to the phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+). VP2 contains basic regions, too. | |
| − | + | <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> | |
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Latest revision as of 16:21, 30 October 2010
pCMV_[AAV2]-VP123
| pCMV_AAV2-VP123 | |
|---|---|
| BioBrick Nr. | BBa_K404007 |
| RFC standard | RFC 10 |
| Requirement | pSB1C3_001 |
| Source | pAAV_RC from Stratagene |
| Submitted by | [http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010] |
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. The VP1 N-terminus contains motifs that are highly homologous to the phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+). VP2 contains basic regions, too.
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), 5178–5189Fitzsimons, 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.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2396
Illegal XhoI site found at 698
Illegal XhoI site found at 884 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 665
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2922
Illegal SapI site found at 1833