Intermediate

Part:BBa_J23068:Design

Designed by: John Anderson   Group: iGEM06_Berkeley   (2006-08-14)


Intermediate #2 in construction of TraGr cassette


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 367
    Illegal suffix found in sequence at 1367
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 367
    Illegal SpeI site found at 1368
    Illegal PstI site found at 1382
    Illegal NotI site found at 373
    Illegal NotI site found at 1375
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 367
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 367
    Illegal suffix found in sequence at 1368
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 367
    Illegal XbaI site found at 382
    Illegal SpeI site found at 1368
    Illegal PstI site found at 1382
    Illegal NgoMIV site found at 300
    Illegal NgoMIV site found at 1429
    Illegal NgoMIV site found at 1635
    Illegal NgoMIV site found at 1653
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1807
    Illegal BsaI site found at 1985
    Illegal BsaI site found at 3053
    Illegal SapI.rc site found at 4135


Design Notes

n/a


Source

PCR JL20/JL21 on RP4 gen. (2268 bp, MfeI/BglII)

Sub into pGLN (BamHI/EcoRI)

Product is pJL20(J23067)

IPCR JL22/JL23 on pJL20(J23067) (3454 bp, EcoRI/PstI)

Sub in pSB1A2-Bca9007 (1015 bp, EcoRI/PstI)

Product is pJL22(J23068)

PCR JL18/JL19 on pJL22(J23068) for D/W (bp, DpnI)

Datsenko Wanner on pKD46/Rlambda,

Product is J23069

JL20 Forward BglII to BamHI for traG-5' knockout cassette

GAGTCAGATCTgattatgttggtgcgctg

JL21 Reverse MfeI to EcoRI for traG-5' knockout cassette

gctggCAATTGcattcgatttccagtacaggc

JL22 Reverse EcoRI IPCR of traG

gctgcGAATTCgatactggaaggaatgggc

JL23 Forward PstI IPCR of traG

cattgCTGCAGacggcagcattgaagaggcg


===References===