pMDS2
(Plasmid #239459)

• Purpose: (Empty Backbone) Simultaneous monitoring of transcription and translation of genes in plant tissues: N- terminus fusion
• Depositing Lab: Miguel de Lucas
• Publication: Banda et al Sci. Adv., 11, eadw9153 (2025)

Backbone

• Vector backbone: pMCY2
• Modifications to backbone: included a 3xHA:VENUS:2A:mTurqN7 module for the simultaneous monitoring of transcription and translation. pMDS2 allows N-ternimal 3xHA:VENUS fusion.
• Vector type: Bacterial Expression, Plant Expression
• Selectable markers: Allows Blue/White selection of promoter fragment insert
• Tag / Fusion Protein:
  • 3xHA:VENUS:2A:mTurqN7 (N terminal on insert)

Growth in Bacteria

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

Cloning Information

• Cloning method: Gibson Cloning

Resource Information

• Supplemental Documents:
  • pMDS2_Addgene.gb

Terms and Licenses

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

Depositor Comments

Two cloning steps are required for the generation of a "promoter:3xHA:VENUS:GENE:2A:mTurqN7" fragment:

A) Cloning of promoter:
Primer design:
Forward Primer: 5’GATCGGGGCGCGCCCTCGAG+PROMOTER SPECIFIC SEQUENCE 3’
Reverse primer: 5’ GATGGGCATTAACATCTTTTAC+PROMOTER SPECIFIC SEQUENCE 3'
Digest plasmid with SapI
Clean
Perform Gibson Assembly
Transform E. coli and select positive colonies LB+Sp/Sm (blue/white selection)

B) Cloning of gene fragment:
Forward Primer: 5’GTTCTGGTGGGGGTGGCTCC+ GENE SPECIFIC SEQUENCE (starting from the ATG) 3’
Reverse primer:  5’ AACAGCTGGCCCGAGCCACC+GENE SPECIFIC SEQUENCE (without STOP Codon) 3'
Digest plasmid with Bsu36I and perform gel purification
Perform Gibson Assembly
Transform E. coli and select positive colonies LB+Sp/Sm

The discrepancies between the depositor's sequence and Addgene's sequences have no functional consequences.