pC-SUR-YR
(Plasmid #61768)

• Purpose: Yeast recombinational cloning compatible Agrobacterium tumefaciens ternary vector containing sulfonylurea resistance gene (ILV alelle from Magnaporthe oyrzae) 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): 12187
• Modifications to backbone: The hygromycin gene in ternary vector pC-HYG-YR (Addgene plasmid# 61765) was replaced with the sulfonylurea (chlorimuron ethyl) resistant gene ILV2SUR (GenBank AF013601), which is a mutated allele of the Magnaporthe oryzae ILV2 gene, to construct pC-SUR-YR.
• Vector type: Ternary vector for Agrobacterium mediated transformation of fungi
• Selectable markers: Sulfonylurea resistance

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: Sur gene
• Alt name: sulfonylurea resistance gene
• Alt name: chlorimuron ethyl resistance gene
• Species: Magnaporthe oryzae
• Insert Size (bp): 2474
• GenBank ID: AF013601.1
• Promoter: Magnaporthe oryzae ILV 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: SUR gene was cloned from pCB1532 (Sweigard et al., 1997)

Terms and Licenses

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

Depositor Comments

SUR gene and its promoter was amplified from cloning vector pCB1532 (Sweigard et al., 1997. Fungal Genetics Newsletter 44: 52-53 http://www.fgsc.net/fgn44/sweig.html) and recombined into pC-HYG-YR (Addgene plasmid# 61765) through yeast recombineering to replace hygromycin gene and create pC-SUR-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 promoter and SUR 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.

Note that Addgene's NGS results identified differences in the SUR gene relative to the Genbank reference sequence AF013601.1, however, the depositor has confirmed that the plasmid functions as described in the associated publication.