Difference between revisions of "Part:BBa K404090"
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<h3>Rep40</h3>
 
<h3>Rep40</h3>
 
{| style="margin: 0px 0px 300px 20px; color: black; float: right;" cellpadding="6" cellspacing="1" border="2"  
 
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! colspan="2" style="background:#66bbff;"|[https://parts.igem.org/Part:BBa_K40490 <i>beta-globin</i> intron]
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! colspan="2" style="background:#009900;"|[https://parts.igem.org/Part:BBa_K40490 rep 40]
 
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! colspan="2"|[[Image:Freiburg10 VectorplasmidBricks 4.png|200px]]
 
! colspan="2"|[[Image:Freiburg10 VectorplasmidBricks 4.png|200px]]
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<p style="margin-right:100px" align="justify">
 
<p style="margin-right:100px" align="justify">
  
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Rep40 in some minutes<br/>
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<span>Rep40 in some minutes<br/>
 
• 40 kDa<br/>
 
• 40 kDa<br/>
 
• ATPase and helicase activity <br/>
 
• ATPase and helicase activity <br/>
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• Involved in genome encapsidation<br/>
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• Involved in genome encapsidation<br/></span>
 
The smallest Rep protein (Rep40) possesses helicase and ATPase activity as well, but does not have strict requirements for DNA duplexes containing a 3´single-stranded end. Rep40 helicase activity requires bivalent ions such as Mg2+ or Mn2+ and is most active using ATP as substrate. Lacking the zinc finger domain, present in Rep52, Rep40 requires dimerization for functional helicase activity (Collaco, Kalman-Maltese, Smith, Dignam, & Trempe, 2003). Rep40/52 proteins are required for translocation of the single-stranded, viral genomes into the preformed capsids proceeding with the 3´end of the DNA (King, Dubielzig, Grimm, & Kleinschmidt, 2001).  
 
The smallest Rep protein (Rep40) possesses helicase and ATPase activity as well, but does not have strict requirements for DNA duplexes containing a 3´single-stranded end. Rep40 helicase activity requires bivalent ions such as Mg2+ or Mn2+ and is most active using ATP as substrate. Lacking the zinc finger domain, present in Rep52, Rep40 requires dimerization for functional helicase activity (Collaco, Kalman-Maltese, Smith, Dignam, & Trempe, 2003). Rep40/52 proteins are required for translocation of the single-stranded, viral genomes into the preformed capsids proceeding with the 3´end of the DNA (King, Dubielzig, Grimm, & Kleinschmidt, 2001).  
  
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<h3>Characterization</h3>
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The BioBrick part beta-globin intron consists partially of a chimeric CMV promoter, followed by the intron II of the beta-globin gene. The 3´end of the intron is fused to the first 20 nucleotides of exon 3 of the beta globin gene. Our BioBrick part beta globin intron is assumed to enhance eukaryotic gene expression. <br />
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AAV-293 cells were transfected with all genes necessary for producing viral particles encapsidating two different rAAV genomes with and without beta-globin intron respectively. mVenus expression was determined by flow cytometry 24-hours post infection of HT1080 cells. The rAAV genomes missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral genomes containing the beta-globin intron. Considering these results, we suggest using the beta-globin intron in dependence on the size of your transgene.
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<h3>References</h3>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 22:36, 26 October 2010

[AAV2]-Rep40ex

Freiburg10 ReplicationBricks 1.png

Rep40

rep 40
Freiburg10 VectorplasmidBricks 4.png
BioBrick Nr. BBa_K40490
RFC standard RFC 10
Requirement pSB1C3
Source pAAV_MCS
Submitted by [http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

Rep40 in some minutes
• 40 kDa
• ATPase and helicase activity
• Involved in genome encapsidation
 The smallest Rep protein (Rep40) possesses helicase and ATPase activity as well, but does not have strict requirements for DNA duplexes containing a 3´single-stranded end. Rep40 helicase activity requires bivalent ions such as Mg2+ or Mn2+ and is most active using ATP as substrate. Lacking the zinc finger domain, present in Rep52, Rep40 requires dimerization for functional helicase activity (Collaco, Kalman-Maltese, Smith, Dignam, & Trempe, 2003). Rep40/52 proteins are required for translocation of the single-stranded, viral genomes into the preformed capsids proceeding with the 3´end of the DNA (King, Dubielzig, Grimm, & Kleinschmidt, 2001).






Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 53
    Illegal XhoI site found at 920
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]