Part:BBa_K176003:Design

lacZalpha-ccdB coding sequence

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 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 SpeI, ligate into pSB1A3.

References

<biblio>

1. You pmid=15064770
2. Balagadde pmid=18414488

</biblio>