Plasmid_Backbone

Part:BBa_K2448036

Designed by: jeremy armetta   Group: iGEM17_Evry_Paris-Saclay   (2017-10-27)


pSB1C3 BsmBI free

This part is the pSB1C3 backbone vector with a single synonymous (G1385C) mutation within the chloramphenicol resistance gene that eliminates the BsmBI cloning site.

Usage and Biology

BsmBI is a type IIS restriction enzyme used in the Golden Gate assembly [1]. Its presence in the backbone of the pSB1C3 plasmid prevents from using this high-throughtput assembly technique with this vector.

For this reason, we decided to perform a site-directed mutagenesis and disrupt the BsmBI recognition site CGTCTC by changing its sequence to CGTGTC. This site is present in the chloramphenicol resistance gene so care was taken that the mutation should not affect the amino acid composition of the protein. The chosen mutation is a synonymous one GTC(Val) to GTG(Val).

                       >BsmBI
                        |
pSB1C3:        1390 - TTC GTC TCA - 1382
                      F   V   S

BBa_K2448036:  1390 - TTC GTG TCA - 1382
                      F   V   S


A relevant application of this part would be the use with it with our Universal Biosensing Chassis (BBa_K2448023 and BBa_K2448024) to build a transcription factor based biosensor finely regulated.


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2049
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 2055
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2049
    Illegal XhoI site found at 1033
    Illegal XhoI site found at 1925
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 2049
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 2049
    Plasmid lacks a suffix.
    Illegal XbaI site found at 2064
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2049
    Illegal XbaI site found at 2064
    Illegal SpeI site found at 2
    Illegal PstI site found at 16


[edit]
Categories
Parameters
None