Part:BBa_K1919001:Design

Constitutive promotor J23105 and downstream CecropinXJ

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 506
  Illegal BamHI site found at 549
  Illegal XhoI site found at 750
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Two steps were needed in order to obtain BBa_K1919001. Step 1, BBa_K1919000 was inserted into pET32a(+). The primers used for PCR are as following:

• F: 5'-CCGGGATCCATGAACTTCGCTAAAATCCTGTCTT-3' (BamH I site was added)
• R: 5'-CCGCTCGAGTTATTATTTACCGATAGCTTTA-3' (Xho I site was added)

BamH I/Xho I Double enzymatic cutting was used for both pET32a(+) and BBa_K1919000.

Step 2, RBS and 5 downstream tags as well as original CecropinXJ was inserted at the downstream of promotor J23105 with the same procedure as step 1. The primers used for PCR are as following:

• F: 5'-GCTCTAGAGCGAAGGAGATATACATATGAGCG-3' (Xba I site was added)
• R: 5'-TGCACTGCAGTGCAGACTAGTCGTTATTGCTCAGCGGTGG-3'(Pst I site was added)

Xba I/Pst I double enzymatic cutting was used for PCR product and Spe I/Pst I double enzymatic cutting was used for BBa_J23105. After enzymatic linkage, the construction of BBa_K1919001 was finished. Then this part was added to pSB1C3 backbone.

Source

The sequence of constitutive promotor J23105 comes from biobrick BBa_J23105. RBS and 5 downstream tags come from commercial plasmid pET32a(+). The sequence of CecropinXJ comes from biobrick BBa_K1919000.