Difference between revisions of "Part:BBa K731700:Design"

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===Design Notes===
 
===Design Notes===
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When using and modifying this construct it is important to remember that the two proteins have strong C-terminal homology. This should be taken into consideration when designing primers. The two proteins have partially overlapping emission and absorption spectra and different folding kinetics.
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When using and modifying this construct it is important to remember that the two proteins have strong C-terminal homology. This should be taken into consideration when designing primers.  
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+
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{|border = "1" cellpadding="5" cellspacing="0" align="left"
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−
|
+
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|Excitation (nm)
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|Emission(nm)
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−
|-
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|mCherry
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|587
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|610
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|-
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|Venus
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|515
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|528
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−
|}
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===Source===
 
===Source===
  
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The part was built starting from a construct kindly provided from the Mansy lab made by Roberta Lentini(RL024). The construct is a pET21b where mCherry and A206K Venus were inserted with a 20 bp spacer between the 2 proteins.
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The part was built starting from RL024A, a construct made by Roberta Lentini and kindly provided by the Mansy lab. The construct is a modified pET21b where mCherry and A206K Venus were inserted. In the starting vector there is also a 20 bp spacer in between the two proteins.
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That plasmid was mutated two times to eliminate illegal restriction sites and a prefix-suffix linker was added by mutagenesis.
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We performed two point mutagenesis and a insertion-deletion mutagenesis on RL024A; in this way we elimitated three illegal restriction sites and inserted the BioBrick cloning region in the spacer's place.
  
 
===References===
 
===References===

Latest revision as of 21:34, 18 September 2012

Platform for terminators analysis under the control of T7 promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 6850
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 6856
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 6850
    Illegal BglII site found at 6011
    Illegal BamHI site found at 6832
    Illegal XhoI site found at 753
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 6850
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 6850
    Plasmid lacks a suffix.
    Illegal XbaI site found at 6865
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 714
    Illegal NgoMIV site found at 1051
    Illegal NgoMIV site found at 4231
    Illegal NgoMIV site found at 4391
    Illegal NgoMIV site found at 5979
    Illegal AgeI site found at 6800
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 2228
    Illegal SapI.rc site found at 3310


Design Notes

When using and modifying this construct it is important to remember that the two proteins have strong C-terminal homology. This should be taken into consideration when designing primers.

Source

The part was built starting from RL024A, a construct made by Roberta Lentini and kindly provided by the Mansy lab. The construct is a modified pET21b where mCherry and A206K Venus were inserted. In the starting vector there is also a 20 bp spacer in between the two proteins. We performed two point mutagenesis and a insertion-deletion mutagenesis on RL024A; in this way we elimitated three illegal restriction sites and inserted the BioBrick cloning region in the spacer's place.

References