Coding

Part:BBa_K929001

Designed by: Potsdam Bioware 2012 iGEM   Group: iGEM12_Potsdam_Bioware   (2012-09-16)
Revision as of 23:33, 25 September 2012 by ChristopherKressler (Talk | contribs)

AID without NES, with NLS and Kozak sequence

AID without NES, with NLS and Kozak sequence
UP12 PlasmidAID without NES, with NLS and Kozak sequence.png
BioBrick Nr. BBa_K929001
RFC standard RFC 10 with aditional AgeI restriction site
Requirement pSB1C3
Source existing part: BBa_K103001
Submitted by [http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]


UP12 PlasmidAID without NES, with NLS and Kozak sequence2.png





















AID - Activation-induced cytidine deaminase

The BioBrick modified AID(BBa_K929001) is an improved version of the existing AID BioBrick (BBa_K103001). It has no NES(Nuclear Export Sequence) sequence and an aditional NLS (Nuclear Localization Sequence) sequence and Kozak sequence added.

The AID motif is naturally terminated with the Nuclear Export Sequence (NES) that causes the protein to translocate from the nucleus to the cytoplasm. Additionally, upstream, dysfunctional Nuclear Localization Sequence (NLS) is located. Due to the fact that AID mutates the actively transcribed single stranded DNA, it is supposed that the direction of the enzyme to the inside of the nucleus would improve the mutation rate.

AID:

AID is known to be responsible for somatic hypermutation and the class-switch recombination of immunoglobulin in B cells. This enzyme of 28 kDa originally occurs in B cells but does also show activity after transfection into CHO cells. AID induces the deamination of cytidine to uridine at actively transcribed single strand DNA. The replacement of cytidine by uridine leads to a mismatch during DNA replication and integrates a single base substitution predominantly in the immunoglobulin genes.

Functional NLS sequence:

This part of the BioBrick directs the expressed protein into the nucleus, where it can mutate stronger.

Kozak sequence

Kozak consensus sequence is added upstream of the AID mutant to express the protein stronger.

Aditional AgeI restriciton site

An aditional AgeI restriciton site was added at the 3` end in front of the stop codon "taa". Therefore fusion with RFC 25 parts at the C-terminus of the modified AID is possible. Incompatibility with RFC 25 is displayed because we defined the AgeI restriction site to be in the insert. Actually because of this AgeI site, fusion with RFC 25 parts is possible.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 579
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 108
    Illegal SapI site found at 209


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Parameters
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