Coding

Part:BBa_K1983006:Design

Designed by: Vykintas Jauniškis   Group: iGEM16_Vilnius-Lithuania   (2016-09-15)


gp45 phenylalanine mutant M37 with C-terminal 6XHis-Tag


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 688
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Designed including NdeI restriction site before the start codon (ATG) for efficient cloning into expression vectors, XhoI site for cutting between the protein and 6XHis-Tag.

Note: Due to technical issues, the part's suffix in the backbone is changed by one FIRST letter (T to G). However, it does not remove or add any other restriction sites and does not change the function of the suffix. The purpose of this note is to alert false negative results during sequencing if the part is used in the future.

Original suffix: TACTAGTAGCGGCCGCTGCAG Part suffix: GACTAGTAGCGGCCGCTGCAG

Gp45M37 mutation list

L220F; L23F; Y172F; I107F; M22F; M187F; Y55F; M194F; L197F; W199F; L167F; Y194F; Y39F; Y216F; M184F; M30F; L10F; I48F; L64F; L127F; L123F; L61F; I54F; I147F; I140F; L160F; I181F; L11F; I83F; I73F; L126F; L57F; I149F; I81F; I17F; I29F; I44F;

Primers

Primers used for amplification of the fragment:
Uni-FW: GTAGAATTCGCGGCCGCTTCTAG
Uni-RV: GTAGACTGCAGCGGCCGCTACTAG

Primers used for colony PCR screening:

For pETDuet-1:
Up-A1: GGATCTCGACGCTCTCCCT
Down3: ACCCCTCAAGACCCGTTTAG

Source

This part is derived T4 phage sliding clamp protein gp45 and was synthesized by Integrate DNA Technologies.

References