pC-HYG-YR
(Plasmid #61765)

• Purpose: Yeast recombinational cloning compatible Agrobacterium tumefaciens ternary vector containing hygromycin 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): 10737
• Modifications to backbone: The Agrobacterium tumfaciens binary vector pCAMBIA-0380 was modified to construct pCHYG (Motteram et al, 2009) by inserting the hygromycin phosphotransferase (hph) gene under the control of Aspergillus nidulans trpC promoter on the TDNA region. A 2µ origin of replication and URA3 gene, from the yeast episomal plasmid YEp24 (Genbank L09156.1), was then inserted at the SacII restriction site in pCHYG to construct yeast recombinational cloning compatible ternary vector pC-HYG-YR.
• Vector type: Ternary vector for Agrobacterium mediated transformation of fungi
• Selectable markers: Hygromycin, 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: Hygromycin
• Species: Synthetic
• 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: The 2 micron origin of replication and URA3 gene were cloned from YEP24 (Genbank L09156.1)
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

Jason Rudd (Rothamsted research UK) provided Agrobacterium tumfaciens vector pCHYG. For more information, see: Motteram et al, 2009. Molecular Plant-Microbe Interactions, 22, 790-799. https://www.ncbi.nlm.nih.gov/pubmed/19522561.

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 hygromycin sequences are present within the EcoRI and HindII cloning sites.

See Supplementary Fig. S1. of associated publication (Sidhu et al., 2015) for schematic of single step assembly of gene deletion ternary vectors using yeast recombinational cloning.