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Reynolds DHFR Saturation Mutagenesis Libraries
(Pooled Library #1000000194, #1000000195, #1000000196)

  • Purpose

    Saturation mutagenesis library of E. coli dihydrofolate reductase (DHFR) in the background of three different thymidylate synthase (TYMS) variants. The TYMS variants are wild-type (WT) E. coli, TYMS Q33S (a moderate loss-of-function mutation), and TYMS R166Q (a catalytically inactive variant).

    Each of the saturation mutagenesis libraries were constructed as four sub-libraries (SL, to allow for coverage of each sub-library region with short read sequencing). SL1 covers all possible single mutations in positions 2–40 (the start codon is unmutagenized), SL2 covers all possible single mutations to positions 41–80, SL3 covers all possible single mutations to positions 81–120, and SL4 covers all possible single mutations to positions 121–159.

  • Vector Backbone

    pTET-Duet - a modified version of the pACYC Duet vector (Novagen), which we edited to introduce a tet repressor upstream of multiple cloning site 2 (in place of the starting T7 promoter)


Item Catalog # Description Quantity Price (USD)
Pooled Library 1000000194 WT TYMS library (four tubes)
  • #191270, SL1 DHFR_WTTS
  • #191271, SL2 DHFR_WTTS
  • #191272, SL3 DHFR_WTTS
  • #191273, SL4 DHFR_WTTS
1 $380 Add to Cart
Pooled Library 1000000195 R166Q TYMS library (four tubes)
  • #191274, SL1 DHFR_R166QTS
  • #191275, SL2 DHFR_R166QTS
  • #191276, SL3 DHFR_R166QTS
  • #191277, SL4 DHFR_R166QTS
1 $380 Add to Cart
Pooled Library 1000000196 Q33S TYMS library (four tubes)
  • #191278, SL1 DHFR_Q33STS
  • #191279, SL2 DHFR_Q33STS
  • #191280, SL3 DHFR_Q33STS
  • #191281, SL4 DHFR_Q33STS
1 $380 Add to Cart
Available to Academic and Nonprofits Only

Library Details

  • Species
  • DHFR variants per library
  • Insert size
    Each plasmid contains DHFR (477 bp), cloned into multiple cloning site 1 and TYMS (792 bp), cloned into multiple cloning site 2.
  • Vector type
    Bacterial expression

Library Shipping

Each library will be delivered as four individual sub-libraries as suspended DNA in microcentrifuge tubes on blue ice. The tube contents will not necessarily be frozen. For best results, minimize freeze-thaws.

  • Volume
    ∼20 µL
  • Concentration
    50 ng/µL

Resource Information

Depositor Comments

Three schematic diagrams showing the plasmid structure for each library (WT TYMS, Q33S TYMS, and R166S TYMS). A promoter, depicted as an arrow, drives expression of DHFR, shown as contiguous rectangular segments. These segments represent the amino acid regions covered in each sub-library. Downstream there is another promoter (arrow) that drives expression of the TYMS variant (wild-type, Q33S, or R166S), which is depicted as a rectangular segment. Each schematic is identical except the relative amino acid position of the TYMS variant is indicated for Q33S and R166Q.
  • Figure 1. DHFR mutagenesis libraries schematic overview. Each sub-library contains all possible single amino acid mutations to DHFR positions 2–40 (SL1), 41–80 (SL2), 81–120 (SL3), and 121–159 (SL4) in one of three TYMS backgrounds (WT, Q33S, or R166Q).

Identification and counting of specific mutants throughout the experiment were performed with the script in GitHub (Link opens in a new window). For a more complete description of the data analysis procedure, please see the methods section of our publication. All major analysis scripts are contained in the accompanying GitHub code repository.

How to cite this pooled library ( Back to top )

These pooled libraries were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    Reynolds DHFR Saturation Mutagenesis library was a gift from Kimberly Reynolds (Addgene #)
  • For your References section:

    The genetic landscape of a metabolic interaction. Nguyen TN, Ingle C, Thompson S, Reynolds KA. Nat Commun. 2024 Apr 18;15(1):3351. doi: 10.1038/s41467-024-47671-0. PubMed 38637543