Protein_Domain
Linker

Part:BBa_K1088051:Design

Designed by: Patrick Rosendahl Andreassen   Group: iGEM13_SDU-Denmark   (2015-03-16)


10 aa linker with BamHI restriction site and TEV recognition site


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Using standard RFC[10] assembly of two parts creates scarsites (tactag). Transcription of the scarsite encodes: "uac uag" of which uag is a stop codon that would render the linker useless. Thus, standard assembly can not be employed for fusing proteins/domains.

Instead we propose the following for implementing the linker:

1) Design primers for amplification of the C-terminal protein/domain that includes the linker DNA sequence in the primer

a) Forward primer: 5'-cgctTCTAGaGGGATCCgaaaatttgtattttcaatctggtNNN...NNN-3' - includes XbaI-site, linker, and sequence complementary to DNA sequence of C-terminal protein/domain

b) Reverse primer: 5'-atatCTGCAGCggccgctACTAGTaNNN...NNN-3' - includes PstI-site, SpeI-site and sequence complementary to DNA sequence of C-terminal protein/domain

2) PCR amplify BioBrick with the designed primers

3) digest pSB1C3 (or any other standard backbone) and amplified PCR product with XbaI and PstI

4) ligate digested pSB1C3 and PCR product to create brick with linker at the N-terminus (pSB1C3-Linker:C-terminal_protein/domain).

5) Design primers for amplification of the N-terminal protein/domain that includes BamHI-site

a) Forward primer: 5'-cgctTCTAGAgNNN...NNN-3' - includes XbaI-site and sequence complementary to DNA sequence of N-terminal protein/domain

b) Reverse primer: 5'-atatGGATCCNNN...NNN-3' - includes BamHI-site and sequence complementary to DNA sequence of N-terminal protein/domain (OBS! do NOT include stop-codons of coding sequences)

6) PCR amplify BioBrick with designed primer

7) digest pSB1C3-Linker:C-terminal_protein/domain and PCR product with XbaI and BamHI

8) ligate the digested pSB1C3-Linker:C-terminal_protein/domain and PCR product to create brick with protein/domain fused with the linker

Source

BBa_K105012

References