Regulatory

Part:BBa_K1603001:Design

Designed by: John Hellgren   Group: iGEM15_Chalmers-Gothenburg   (2015-09-18)
Revision as of 13:28, 18 September 2015 by Jannie (Talk | contribs) (→‎Design Notes)


pTEF1-pSUC2


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 167


Design Notes

Added 20 overlapping base pairs between the two gene fragments through PCR with primers with overhangs. This allowed the two promoters to be combined through Gibson assembly. Added Prefix and Suffix through PCR with primers with overhangs.


Primers for isolation of pTEF1 with Prefix from genomic DNA:

FW:GCTTCTAGATAGCTTCAAAATGTTTCTACTCCTTTT

RV:TTTGTAATTAAAACTTAGATTAGATTGCTATGCT


Primers for isolation of pSUC2 with overlap to pTEF1 and Suffix from genomic DNA:

FW:ATCTAAGTTTTAATTACAAAATAAATAGATATGTATTATTCTTCAAAACATTCTC

RV:AGCCTGCAGCGGCCGCTACTAGTAATACGTTAGTGAAAAGAAAAGCTTTTTG


The primers were designed to remove the EcoRI site from the insert when constructing the biobrick to reduce the amount of base pairs in the primers. The primers also add 3 extra protective basepairs to the PstI site in the insert.


The construction of the final biobrick was initiated by cutting the vector (BBa_J04450) with XbaI, PstI, KnpI and FastAP and the insert with XbaI and PstI.

Source

Genomic sequence from Saccharomyces cerevisiae CEN.PK2

References