KG#901
(Plasmid #110936)

• Purpose: Expresses Emerald-RAB-3 in the cholinergic motor neuron DA9 for imaging synaptic vesicles in a single neuron in a living animal
• Depositing Lab: Kenneth Miller
• Publication: Edwards et al Genetics. 2018 Nov;210(3):947-968. doi: 10.1534/genetics.118.301466.

Backbone

• Vector backbone: pUC
• Backbone size w/o insert (bp): 2632
• Total vector size (bp): 8455
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: mig-13 promoter
• Species: C. elegans (nematode)
• Insert Size (bp): 3393

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: Unknown

Gene/Insert 2

• Gene/Insert name: Emerald-RAB-3
• Species: C. elegans (nematode)
• Insert Size (bp): 1395

Cloning Information for Gene/Insert 2

• Cloning method: Unknown
• 5′ sequencing primer: Unknown

Gene/Insert 3

• Gene/Insert name: unc-54 3' control region with 1 artificial intron just upstream and 1 artificial intron in control region
• Species: C. elegans (nematode)
• Insert Size (bp): 940

Cloning Information for Gene/Insert 3

• Cloning method: Unknown
• 5′ sequencing primer: Unknown

Resource Information

• Supplemental Documents:
  • Annotated ApE and Strider formatted file--download free ApE plasmid editor to view

Terms and Licenses

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

Depositor Comments

Used ApE plasmid editor to digitally construct the plasmid, then designed the primers necessary to assemble Emerald and RAB-3 into the mig-13:: vector background. We then used Herculase II to produce the fragments and the NEBuilder Master Mix to make the new plasmid. Transformed into XL1-Blue electrocompetent cells. Did Qiagen preps on 2 clones, screened by PCR, and submitted 1 clone for sequence of the Emerald-RAB-3 region. Froze the DNA and made a glycerol stock.

Sizes and locations of the component fragments the below table:

KG#471 mig-13 promoter and vector sequences 7.1 kb
KG#858 Emerald 720 bp
KG#776 rab-3 cDNA 660 bp

In transgenic animals, this vector will produce Emerald-RAB-3 to tag synaptic vesicles in the DA9 cholinergic motor neuron, thus allowing visualization of synaptic vesicles in a single neuron in living animals.
Features of the expression construct: The mig-13 promoter drives expression in DA9 from hatching to adult and VA12 from mid-larval to adult stages. Also drives expression occasionally in PHAL, PVCL, and LUAL in addition to many neurons anterior to the vulva. This vector has 3 synthetic introns for increasing the robustness of intronless insert expression: 1 in the 5' UTR, one just upstream of the unc-54 3' UTR/ control region, and one in the 3' UTR of unc-54. The new MCS in this construct is derived from pPD96.52, with the addition of an 8-cutter Sbf I site between Nhe I and Kpn I.

Note: this vector can be converted to a vector that fuses GFP, YFP, or CFP onto the C-terminus of the protein as follows:

Remove the ~1000 bp (Kpn I or Age I) + (Spe I or BsiW I [expensive, 55 C cutter with 50% activity at 37 C; heat inactivate 80 C] or Apa I) fragment containing the unc-54 3’ control region from this plasmid and replace it with the ~1800 bp like-digested fragment containing 3-intron S65C GFP (see pPD94.81 in Fire Lab plasmids), YFP (see pPD136.64 in Fire Lab plasmids) or CFP (see pPD136.61 in Fire Lab plasmids) + the unc-54 3’ UTR and control region. Note if Spe I is used to cut these Fire Lab vectors, you get 3 bands: 1100, 1800, and 4000 (total size 6.9 Kb). The 1800 bp band is the XFP-containing band.

For C-terminal fusions, remember to leave the stop codon off of the upstream protein. For the above-mentioned Fire Lab vectors, the reading frame for the downstream XFP relative to the Kpn I and Age I sites is as follows (triplets are the reading frame codons):

Kpn I
G GTA CCG GT
Age I

Alternatively, use Gibson Assembly/ NEBuilder to insert any genetically encoded fluorescent protein at the N- or C-terminus of your protein.