Difference between revisions of "Part:BBa J176171"
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<partinfo>BBa_J176171 short</partinfo>
 
<partinfo>BBa_J176171 short</partinfo>
  
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Mammalian expression vector (without a promoter) designed to add a eukaryotic RBS (Kozak) upstream (in-frame) and a nuclear localization tag, 6-histidine tag, and stop codon downstream (in-frame) of a protein coding insert. This method only works for Silver Standard (RFC23) inserts.
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Mammalian expression vector (without a promoter) designed to add a eukaryotic RBS (Kozak) upstream (in-frame) and a nuclear localization tag (NLS), 6-histidine tag, and stop codon downstream (in-frame) of a protein coding insert. This method only works for Silver Standard (RFC23) inserts.<br>
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* Alias: MV9
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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'''A Promoter Must be Added for Expression'''
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* This vector has no promoter. This design allows the user to customize expression.
 +
* We suggest cutting this vector with SpeI/PstI and inserting a XbaI/PstI-cut promoter (insert).
 +
* Alternatively, the multiple cloning site allows different strategies for inserting a promoter upstream of the Kozak sequence.
 +
 +
'''Insert a Protein-coding Sequence'''
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* Cut this vector with XbaI. This will open an insertion site between the Kozak and the NLS.
 +
* Dephosphorylate (phosphatase-treat) the vector to prevent self re-ligation.
 +
* Cut your protein-coding insert with XbaI/SpeI.
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** The insert must have its XbaI and SpeI sites immediately adjacent to the protein coding sequence.
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** The insert must '''not''' have a Kozak (RBS) or stop codon.
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* Ligate the XbaI/SpeI-cut insert into the XbaI-cut, dephosphorylated vector.
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* Pick at least three colonies to increase the likelihood of a forward insert. Since all sticky ends are compatible, the insert can ligate in either direction (forward or reverse). Only forward inserts will express properly.
 +
 +
'''Should the Promoter be Added First or Second?'''
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* If you want to build a set of plasmids that express different proteins, under control of the same type of promoter, build the Promoter+vector plasmid first
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* If you want to build a set of plasmids that express a single protein, under the control of different types of promoters, build the Protein-coding sequence+vector plasmid first
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Revision as of 18:18, 22 April 2013

Kozak-XbaI-NLS-6His-stop

Mammalian expression vector (without a promoter) designed to add a eukaryotic RBS (Kozak) upstream (in-frame) and a nuclear localization tag (NLS), 6-histidine tag, and stop codon downstream (in-frame) of a protein coding insert. This method only works for Silver Standard (RFC23) inserts.

  • Alias: MV9


Usage and Biology

A Promoter Must be Added for Expression

  • This vector has no promoter. This design allows the user to customize expression.
  • We suggest cutting this vector with SpeI/PstI and inserting a XbaI/PstI-cut promoter (insert).
  • Alternatively, the multiple cloning site allows different strategies for inserting a promoter upstream of the Kozak sequence.

Insert a Protein-coding Sequence

  • Cut this vector with XbaI. This will open an insertion site between the Kozak and the NLS.
  • Dephosphorylate (phosphatase-treat) the vector to prevent self re-ligation.
  • Cut your protein-coding insert with XbaI/SpeI.
    • The insert must have its XbaI and SpeI sites immediately adjacent to the protein coding sequence.
    • The insert must not have a Kozak (RBS) or stop codon.
  • Ligate the XbaI/SpeI-cut insert into the XbaI-cut, dephosphorylated vector.
  • Pick at least three colonies to increase the likelihood of a forward insert. Since all sticky ends are compatible, the insert can ligate in either direction (forward or reverse). Only forward inserts will express properly.

Should the Promoter be Added First or Second?

  • If you want to build a set of plasmids that express different proteins, under control of the same type of promoter, build the Promoter+vector plasmid first
  • If you want to build a set of plasmids that express a single protein, under the control of different types of promoters, build the Protein-coding sequence+vector plasmid first


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 266
    Illegal XbaI site found at 329
    Illegal SpeI site found at 249
    Illegal PstI site found at 271
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 266
    Illegal SpeI site found at 249
    Illegal PstI site found at 271
    Illegal NotI site found at 292
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 266
    Illegal BglII site found at 12
    Illegal XhoI site found at 299
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 266
    Illegal XbaI site found at 329
    Illegal SpeI site found at 249
    Illegal PstI site found at 271
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 266
    Illegal XbaI site found at 329
    Illegal SpeI site found at 249
    Illegal PstI site found at 271
    Illegal NgoMIV site found at 819
    Illegal NgoMIV site found at 2230
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 1955
    Illegal BsaI.rc site found at 3755
    Illegal SapI site found at 2675