Difference between revisions of "Part:BBa K1603001:Design"
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Primers for isolation of pTEF1 with Prefix and overlap to pSUC2 from genomic DNA: | Primers for isolation of pTEF1 with Prefix and overlap to pSUC2 from genomic DNA: | ||
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FW:GCTTCTAGATAGCTTCAAAATGTTTCTACTCCTTTT | FW:GCTTCTAGATAGCTTCAAAATGTTTCTACTCCTTTT | ||
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Primers for isolation of pSUC2 with overlap to pTEF1 and Suffix from genomic DNA: | Primers for isolation of pSUC2 with overlap to pTEF1 and Suffix from genomic DNA: | ||
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FW:ATCTAAGTTTTAATTACAAAATAAATAGATATGTATTATTCTTCAAAACATTCTC | FW:ATCTAAGTTTTAATTACAAAATAAATAGATATGTATTATTCTTCAAAACATTCTC | ||
Revision as of 12:12, 18 September 2015
pTEF1-pSUC2
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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 and overlap to pSUC2 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 EcoRI site was removed from the insert when constructing the biobrick to reduce the amount of base pairs in the primers. 3 extra protective basepairs were added to the PstI site.
The construction of the final biobrick product 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