Signalling

Part:BBa_I756028:Design

Designed by: Jennifer Lei   Group: iGEM07_Bay_Area_RSI   (2007-10-25)


cmv - IgM signal- VH-VL-EporD2-EporTm mut- gp130i- dnaEC-VP16-SV40PA


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1
    Illegal XbaI site found at 7
    Illegal SpeI site found at 3033
    Illegal PstI site found at 2299
    Illegal PstI site found at 2460
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal NheI site found at 2704
    Illegal SpeI site found at 3033
    Illegal PstI site found at 2299
    Illegal PstI site found at 2460
    Illegal NotI site found at 15
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
    Illegal BglII site found at 546
    Illegal BglII site found at 1963
    Illegal BglII site found at 2227
    Illegal BamHI site found at 1212
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1
    Illegal XbaI site found at 7
    Illegal SpeI site found at 3033
    Illegal PstI site found at 2299
    Illegal PstI site found at 2460
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1
    Illegal XbaI site found at 7
    Illegal SpeI site found at 3033
    Illegal PstI site found at 2299
    Illegal PstI site found at 2460
    Illegal NgoMIV site found at 1514
    Illegal NgoMIV site found at 2609
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1776
    Illegal SapI site found at 1041


Design Notes

EporD2 has >70% content and is extremely difficult to clone.


Source

EcoR1,Xba1,SacII (1-18bp) cmv from pcDNA3.1 zeo(+)(19-550bp) bstB1 (551-556bp) kozak (557-562bp) IgM signal sequence from IgM of mouse (made synthetically from oligos) (563-619bp)

Variable heavy chain of human IgM monoclonal AB (EMBL: AJ224899) and Variable light (EMBL: AJ224900) sequence from Laroche, FEBS Letter, 1999;460, 86-92 (made synthetically from oligos) (620-1384bp)

EporD2 sequence from Kawahara, M et al. Protein Engineering Design and Selection. v.17, pp.715-719, 2004 ( isoalted from human NT2 cDNA) (1385-1708bp)

EporTM mut sequence from Kawahara,M et al. Protein Engineering Design and Selection. v.17, pp.715-719, 2004 ( made synthetically from oligos) (1709-1768bp)

GP130i sequence from Kawahara, M et al. Protein Engineering Design and Selection. v.17, pp.715-719, 2004 (isolated from human NT2 cDNA)(1769-2599bp)

Linker (2600-2623bp)

dnaEC from dnaE gene of Synechocystis sp.(split inteins)(2624-2749bp)

VP16 from HSV1 helper virus (Sapolsky Lab, Stanford)(2750-2887bp) Stop Stop (2888-2893bp) SV40 polyA from pcDNA3.1 zeo(+)(2894-3024bp)

Asc1,Spe1 (3025-3038bp)




References