Part:BBa_K4047016

Gentamicin resistance (GmR) for Synechococcus

This part is the coding sequence conferring gentamicin resistance, used within the plasmid BBa_K4047039. This plasmid deletes a target gene by insertion of kanamycin BBa_K4047019. This part encodes gentamicin resistance (GmR) in the plasmid backbone.

This marker is used to differentiate single versus double crossover events in order to identify successful deletion mutants. Successful transformants only integrate kanamycin in order to delete of the target gene glpX. Incorrect crossover leads to integration of both kanamycin and gentamicin resistance genes, and allows functional expression of glpX. Thus, the two genes in conjunction allow selection for glpX deletion by selecting those mutants which can only grow on kanamycin and not gentamicin. Although gentamicin resistance indicates failure of target gene deletion, it is extremely useful as a marker to differentiate transformants.

This gene was functionally expressed and conferred antibiotic resistance, as some transformed colonies were able to grow on both kanamycin and gentamicin, indicating the successful expression and operation of this gene. See the Experience tab for more details on the successful operation of this part in the MiamiU_OH project.

This part is only the coding region for gentamicin resistance, with the promoter BBa_K4047015 and terminator BBa_K4047017 being vital accessory components to its functional operation.

This part is optimized for operation in Synechococcus elongatus PCC 7942, exhibiting gentamicin resistance for low concentrations. This sequence varies from the established gentamicin resistance part BBa_P1014, which conveys resistance to gentamicin at up to 12 ug/ml in E. coli.

The marker shares 100% nucleotide identity with Shuttle vector pXsp8, found at the following Genbank link (https://www.ncbi.nlm.nih.gov/nucleotide/KC817027.1?report=genbank&log$=nuclalign&blast_rank=1&RID=R0SPGK2Z013)

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 314
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Long Description

Sequence encoding gentamicin resistance (gmr gene)