pC-G418-YR
(Plasmid #61767)

• Purpose: Yeast recombinational cloning compatible Agrobacterium tumefaciens ternary vector containing nptII (neomycin phosphotransferase II) selectable marker on transfer DNA (TDNA).
• Depositing Lab: Ken Haynes
• Publication: Sidhu et al Fungal Genet Biol. 2015 Jun;79:102-9. doi: 10.1016/j.fgb.2015.04.015.

Backbone

• Vector backbone: pCAMBIA-0380
• Backbone manufacturer: CAMBIA
• Backbone size w/o insert (bp): 6812
• Total vector size (bp): 10768
• Modifications to backbone: The hygromycin gene in ternary vector pC-HYG-YR (Addgene plasmid# 61765) was replaced with nptII gene (Geneticin/G418 resistance) to construct pC-G418-YR.
• Vector type: Ternary vector for Agrobacterium mediated transformation of fungi
• Selectable markers: Neomycin (select with G418), URA3

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: nptII gene
• Alt name: Geneticin or G418 resistance gene
• Species: E. coli
• Insert Size (bp): 795
• GenBank ID:
• Promoter: trpC promoter

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: EcoRI (not destroyed)
• 3′ cloning site: HindIII (not destroyed)
• 5′ sequencing primer: LB-F1 (5'-gtggtgtaaacaaattgacgc-3')
• 3′ sequencing primer: RB-F1 (5'-ggataaaccttttcacgccc-3')

Gene/Insert 2

• Gene/Insert name: 2 micron origin or replication and URA3 gene for S. cerevisiae
• Species: S. cerevisiae (budding yeast)
• Insert Size (bp): 2487
• GenBank ID: KC562906.1

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: SacII (not destroyed)
• 3′ cloning site: SacII (not destroyed)
• 5′ sequencing primer: ccgcggTTAGTTTTGCTGGCCGCATC
• 3′ sequencing primer: ccgcggCGCTTCCACAAACATTGCTC

Resource Information

• Supplemental Documents:
  • Annotated GenBank file
• A portion of this plasmid was derived from a plasmid made by: nptII gene (Geneticin/G418 resistance) was cloned from pCGEN

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Jason Rudd, (Rothamsted research UK) provided Agrobacterium tumfaciens vector pCGEN (pCAMBIA-0380 backbone with nptII gene under Neurospora crassa gpd promoter).

NptII (neomycin phosphotransferase II) gene from pCGEN was recombined into pC-HYG-YR (Addgene plasmid# 61765) through yeast recombineering to replace hygromycin gene and create pC-G418-YR.

Restriction sites EcoRI and HindIII can be used to linearise the vector inside left (LB) and right (RB) borders of the TDNA. Note that the trpC promoter and nptII sequences are present within the EcoRI and HindII cloning sites.

See Supplementary Fig. S1. of associated publication for schematic of single step assembly of gene deletion ternary vectors using yeast recombinational cloning.

Please note that the Addgene verified sequence identified a transposase in the backbone (7145-8353 bp).