Part:BBa_K341456

Kanamycin resistance(with Promoter) + I-sceI site+ Recombination site

Kanamycin resistance(with Promoter) + I-sceI site+ Recombination site+DraIII cutting site. This part can be used as a recombination unit between a plasmid and the chromosome in the "In-vivo Recombination System" in iGEM project "Tsinghua 2010"

This part involves DraIII cutting site at two ends of the unit, this cutting site is compatible with BBF RFC 61

Thsi part is uesd as a Insertion Fragment of Donor Plasmid in the In-vivo Recombination System of iGEM Tsinghua 2010 Project.

Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:

1) one 15bp-long restriction enzyme I-scel recognition site

2) one 25bp-long random sequence

3) one fragment for insertion and recombination

4) one 25bp-long random sequence

5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)

Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.

In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 893
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 229
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 169
  Illegal SapI site found at 379
  Illegal SapI site found at 1225
  

Safety

This part is for research use only, no special safety issues need to be mentioned.

References

Thomas E. Kuhlman and Edward C. Cox:Site-specific chromosomal integration of large synthetic constructs,Nucleic Acids Research, 2009, 1–10

Acknowledgement

The physical DNA is from Vecter "pTKs-CS" which is generously provided by Thomas E. Kuhlman and Edward C. Cox of the Department of Molecular Biology, Princeton University.

We are grateful to Guoqiang Chen, Zhao Wang,Yinghua Chen and Liping Xie of School of Life Sciences, Tsinghua University for assistance in the design, construction and characterization of this part.