Difference between revisions of "Part:BBa K176003:Design"
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Reverse Primer: GTTTCTTC CTGCAG CGGCCGC T ACTAGT A TTATTA tattccccagaacatcaggtta | Reverse Primer: GTTTCTTC CTGCAG CGGCCGC T ACTAGT A TTATTA tattccccagaacatcaggtta | ||
| − | Digest with XbaI and | + | Digest with XbaI and PstI, ligate into pSB1A3. |
===References=== | ===References=== | ||
Latest revision as of 12:34, 14 September 2009
lacZalpha-ccdB coding sequence
Assembly Compatibility:
- 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 site found at 387
Design Notes
The part is similar to BBa_J32026 and the original coding sequence in pluxCcdB3, but the multiple cloning sites are eliminated in order to avoid site-specific mutagenesis. The multiple cloning sites are from lacZalpha in pZErO-2.
After eliminate the MCS, the lacZalpha part is identical to the N-terminal of the wild type gene(BBa_I732006 & BBa_I732005), and fused with the ccdB part (BBa_K145151) with a 3aa linker.
Source
PCR from the plasmid pluxCcdB3You Balagadde (a gift from Prof. Lingchong You).
Forward Primer: GTTTCT TCTAG ATG accatgattacgg attcactggccgtcgttttac
Reverse Primer: GTTTCTTC CTGCAG CGGCCGC T ACTAGT A TTATTA tattccccagaacatcaggtta
Digest with XbaI and PstI, ligate into pSB1A3.
References
<biblio>
- You pmid=15064770
- Balagadde pmid=18414488
</biblio>