Difference between revisions of "Part:BBa K404009"
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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. 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. | 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. | ||
| − | + | <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> | <h3>References</h3> | ||
<b>Bleker, Pawlita, & Kleinschmidt</b>, 2006. Impact of capsid conformation and rep-capsid interactions on adeno-associated virus type 2 genome packaging. Journal of Virology, 810–820 <br /> | <b>Bleker, Pawlita, & Kleinschmidt</b>, 2006. Impact of capsid conformation and rep-capsid interactions on adeno-associated virus type 2 genome packaging. Journal of Virology, 810–820 <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 <br /> | ||
<center><img src="https://static.igem.org/mediawiki/parts/a/a7/Freiburg10_Cap_proteins_VP1_2%263.png" width="600" | <center><img src="https://static.igem.org/mediawiki/parts/a/a7/Freiburg10_Cap_proteins_VP1_2%263.png" width="600" | ||
height="auto" margin: 10px 10px 10px 10px/></center> | height="auto" margin: 10px 10px 10px 10px/></center> | ||
Latest revision as of 16:28, 30 October 2010
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, 810–820Fitzsimons, 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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 701
Illegal XhoI site found at 887 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 665
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1836