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Species: Caenorhabditis elegans
Genetic Insert: rab-3 promoter
Vector Backbone Description: Backbone Size:2617; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15489511
Comments: Always streak out this culture for single colonies and do minipreps on >= 4 clones (~1/2 of colonies will be right). Choose small colonies after 18 - 20h growth. DNA yeild is low (~170 ng/ ul x 30 ul for a Qiagen 3 ml miniprep).
Used Pfu Ultra polymerase and primers engineered with restriction sites to amplify the 3.8 Kb acy-1 cDNA coding region from KG#61 and cloned into Age I/ Xho I cut KG#59 (rab-3:: expression vector). Transform into XL1-Blue electrocompetent cells. Miniprepped 8 clones to find those with correct size insert and vector, then submitted 2 clones for sequence. Chose one clone that has correct sequence and made glycerol stock.
Features of the expression construct: This expression construct has a promoter (rab-3) that will drive strong and specific expression of acy-1 cDNA in the entire nervous system of juvenile and adult animals. Other features of the expression vector include a "decoy" 5' to the promoter to reduce non-specific expression in the gut (see Fire Lab 1995 Kit documentation for description), the unc-54 3' end including the poly A addition signal, and 3 introns (1 in 5' UTR, 1 just upstream of the unc-54 3' end, and 1 inserted in the unc-54 3' end). These introns help provide more uniform and robust expression, especially if you are expressing a cDNA with no introns (as we are) as opposed to a gene with introns. The sequence AAAA is engineered just after the 5' restriction site and just before the initiator ATG to provide a consensus translation start site. The sequence AT follows the stop codon and immediately precedes the 3' restriction site to provide a consensus post - stop codon sequence. The vector provides the unc-54 3' UTR and control region. Note: the acy-1 cDNA from which this clone was produced corresponds to the single acy-1 cDNA predicted by wormbase, which is F17C8.1
Proper citation: RRID:Addgene_110874 Copy
Species: Caenorhabditis elegans
Genetic Insert: rab-3 promoter
Vector Backbone Description: Backbone Size:2617; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16272411
Comments: Used Pfu Ultra polymerase and primers engineered with restriction sites to amplify the 3780 bp unc-31 cDNA coding region from KG#104 and cloned into Nhe I/ Age I cut KG#59 (rab-3:: expression vector). Transformed into XL1-Blue electrocompetent cells. Miniprepped 6 clones to find those with correct size insert and vector, then submitted 2 clones for sequence. Chose one clone that has correct sequence and made glycerol stock.
Features of the expression construct: This expression construct has a promoter (rab-3) that will drive strong and specific expression of unc-31 cDNA in the entire nervous system of juvenile and adult animals. Other features of the expression vector include a "decoy" 5' to the promoter to reduce non-specific expression in the gut (see Fire Lab 1995 Kit documentation for description), the unc-54 3' end including the poly A addition signal, and 3 introns (1 in 5' UTR, 1 just upstream of the unc-54 3' end, and 1 inserted in the unc-54 3' end). These introns help provide more uniform and robust expression, especially if you are expressing a cDNA with no introns (as we are) as opposed to a gene with introns. The sequence AAAA is engineered just after the 5' restriction site and just before the initiator ATG to provide a consensus translation start site. The sequence AT follows the stop codon and immediately precedes the 3' restriction site to provide a consensus stop translation signal. The vector provides the unc-54 3' UTR and control region.
Note: this unc-31 cDNA from lacks the first 408 bp of the full-length unc-31 cDNA, but confers full rescue activity for locomotion.
Proper citation: RRID:Addgene_110879 Copy
Species: Caenorhabditis elegans
Genetic Insert: mig-13 promoter
Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:26354975
Comments: Unique multi-cloning sites: Asc I [left of 5’ UTR] / Sbf I/ Kpn I/ EcoRV/ Xho I
Used BamH I/ Apa I to cut out the ~825 bp MCS + unc-54 3' control region from KS#1 mig-13:: expression vector (gift of Kang Shen) , leaving the 6.1 kb vector fragment containing the mig-13:: promoter. To this vector fragment, we ligated the 1000 bp BamH I/ Apa I fragment from KG#470. Do Earth minipreps on 2 colonies. Chose 1 clone with correct size fragments and made a glycerol stock.
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.
Proper citation: RRID:Addgene_110935 Copy
Species: Caenorhabditis elegans
Genetic Insert: unc-17 beta promoter
Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16272411
Comments: Unique multi-cloning sites: Nhe I, Sal I, Acc I, Kpn I/ Age I/ Eco RV, Xho I, Sac I, Bgl II
Used Pfu Ultra polymerase and primers engineered with restriction sites to amplify the 0.5 Kb unc-17 beta promoter from RM#605p DNA (gift of Jim Rand) and cloned into Pst I/ Bam HI cut pPD96.52 (C. elegans body wall muscle expression vector; this digestion will remove the myo-3 promoter in 2 fragments and leave the 3.7 kb vector). Transformed into XL1-Blue electrocompetent cells. Miniprepped 6 clones to find those with correct size insert and vector, then submitted 2 clones for sequence. Chose one clone that has correct sequence and made a glycerol stock.
Features of the expression construct: This expression construct has a promoter (unc-17 beta) that will drive strong and specific expression (of whatever gene is inserted in the MCS) in the A and B class motor neurons and the AS cells of the ventral nerve cord of juvenile and adult animals (does not drive expression in VC's, but still gives full behavioral rescue of unc-17 beta mutants (by thrashing assay). The promoter is an artificial fusion of the unc-17 promoter beta region (115 bp Hind III - Xba I fragment with the unc-17 basal promoter region (378 bp region from Nar I to middle of the non-coding exon 1 of cha-1/ unc-17. Other features of the expression vector include a "decoy" 5' to the promoter to reduce non-specific expression in the gut (see Fire Lab 1995 Kit documentation for description), the unc-54 3' end including the poly A addition signal, and 3 introns (1 in 5' UTR, 1 just upstream of the unc-54 3' end, and 1 inserted in the unc-54 3' end). These introns help provide more uniform and robust expression, especially if you are expressing a cDNA with no introns as opposed to a gene with introns.
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.
Proper citation: RRID:Addgene_110933 Copy
Species: Caenorhabditis elegans
Genetic Insert: mig-13 promoter
Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30401765
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.
Proper citation: RRID:Addgene_110936 Copy
Species: Caenorhabditis elegans
Genetic Insert: mig-13 promoter
Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30401765
Comments: Used ApE plasmid editor to digitally construct the plasmid, then designed the primers necessary to assemble INS-22-Emerald 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 INS-22-emerald region. Froze the DNA and made a glycerol stock.
In transgenic animals, this vector will produce INS-22-Emerald to tag Dense Core Vesicles in the DA9 cholinergic motor neuron, thus allowing visualization of Dense Core 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.
Proper citation: RRID:Addgene_110937 Copy
Species: Caenorhabditis elegans
Genetic Insert: hsp-16-2 promoter
Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15489511
Comments: Unique sites in multi-cloning site: Nhe I/ Kpn I/ Age I/ Eco RV/ Xho I/ Sac I/ Bgl II
We used Hind III/ Bam HI to cut out the 2410 bp myo-3 promoter from pPD96.52 (Fire Lab plasmid), leaving the 3684 bp vector fragment. To this vector fragment, we ligated the ~430 bp hsp16-2 promoter Hind III/ Bam HI fragment cut from pPD49.78 (Fire Lab plasmid).
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.
Proper citation: RRID:Addgene_110926 Copy
Species: Caenorhabditis elegans
Genetic Insert: unc-129 promoter
Vector Backbone Description: Backbone Size:2617; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30401765
Comments: We used Herculase II DNA polymerase and primers engineered with restriction sites to amplify and clone the 1.4 Kb strn-1 (C16E9.2) cDNA (minus its stop codon) from KG#720 rab-3::strn-1-FLAG into Nhe I/ Kpn I cut KG#367 (unc-129::___-GFP expression vector; 7.2 Kb) such that it is in-frame with the GFP. Transformed into XL1-Blue electrocompetent cells. Did Qiagen preps on 2 clones to find one with correct size insert for sequence verification. Verified sequence.
Features of the construct: The unc-129:: promoter drives expression in a subset of 9 DA/ DB cholinergic motor neurons. AAAA precedes the start codon to provide a consensus ribosome binding site. A non-structured linker (SSGSSG) replaces the strn-1 stop codon and is immediately followed by the Fire vector GFP with 3 artificial introns (there are also introns in the 5' and 3' UTR in this vector). 2 bases are added to maintain the reading frame with the downstream GFP when cloning into the Kpn I site (this encodes an Ala).
Proper citation: RRID:Addgene_110916 Copy
Species: Caenorhabditis elegans
Genetic Insert: unc54 Terminator
Vector Backbone Description: Backbone Marker:Mendenhall Lab; Vector Backbone:BSP179; Vector Types:Worm Expression, Synthetic Biology, Yeast Homologous Recombination cloning for Universal MosSCI; Bacterial Resistance:Kanamycin
Defining Citation: PMID:33693300
Proper citation: RRID:Addgene_110917 Copy
Species: Caenorhabditis elegans
Genetic Insert: ACid Phosphatase family 2
Vector Backbone Description: Vector Backbone:pCI-neo; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23233530
Proper citation: RRID:Addgene_113051 Copy
Species: Caenorhabditis elegans
Genetic Insert: MiRP K+ channel accessory Subunit 1 1
Vector Backbone Description: Vector Backbone:pCI-neo; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:12533541
Proper citation: RRID:Addgene_113057 Copy
Species: Caenorhabditis elegans
Genetic Insert: K+ channel for Habituation to Tap subunit 1
Vector Backbone Description: Vector Backbone:pCI-neo; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:19387491
Proper citation: RRID:Addgene_113054 Copy
Species: Caenorhabditis elegans
Genetic Insert: MiRP K+ channel accessory Subunit 1
Vector Backbone Description: Vector Backbone:pCI-neo; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:17604313
Comments: This plasmid contains an N214K mutation compared to the reference sequence NP_495263.2 that is not of concern for function.
Proper citation: RRID:Addgene_113059 Copy
Species: Caenorhabditis elegans
Genetic Insert: K+ voltage sensitive channel subunit 1.2
Vector Backbone Description: Vector Backbone:pCI-neo; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:17488718
Comments: This plasmid contains K681I and T208A mutations in KVS-1 compared the reference AAO65852.1 that do not impact function.
Proper citation: RRID:Addgene_113069 Copy
Species: Caenorhabditis elegans
Genetic Insert: lis-1
Vector Backbone Description: Backbone Marker:L4440 is available at Addgene; Backbone Size:2790; Vector Backbone:L4440; Vector Types:Worm Expression, RNAi; Bacterial Resistance:Ampicillin
Comments: Full length cDNA for C. elegans open-reading frame T03F6.5, encoding the lis-1 gene, cloned into RNAi feeding vector L4440. Can be used for RNAi feeding; should result in embryonic lethality at high levels of induction.
This plasmid is intended for use exclusively as a teaching resource as part of the Integrated Genomics Discovery-Based Laboratory Course. Addgene does not make any guarantee that the plasmid is suitable for research purposes.
Proper citation: RRID:Addgene_11336 Copy
Species: Caenorhabditis elegans
Genetic Insert: lin-41 miRNA target sequence mut
Vector Backbone Description: Backbone Marker:Available at Addgene (plasmid 12178); Backbone Size:5264; Vector Backbone:pIS0; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:14697198
Comments: Used as a control for testing effects of lin-41 microRNA (let-7).
Proper citation: RRID:Addgene_11907 Copy
Species: Caenorhabditis elegans
Genetic Insert: TagRFP-T^SEC^BioTag::AID*::3xFlag
Vector Backbone Description: Backbone Size:2600; Vector Backbone:pUC19 (modified); Vector Types:Worm Expression, Cre/Lox, CRISPR; Bacterial Resistance:Ampicillin
Defining Citation: PMID:33677541
Comments: This plasmid is derived from pDD284 (AddGene plasmid 66825)
Please note: This plasmid contains an eight basepair deletion at end of the homology arm near M13-rev. This deletion is not known to affect plasmid function.
Proper citation: RRID:Addgene_121061 Copy
Species: Caenorhabditis elegans
Genetic Insert: mKate2^SEC^BioTag::AID*::3xFlag
Vector Backbone Description: Backbone Size:2600; Vector Backbone:pUC19 (modified); Vector Types:Worm Expression, Cre/Lox, CRISPR; Bacterial Resistance:Ampicillin
Defining Citation: PMID:33677541
Comments: This plasmid is derived from pDD285 (AddGene plasmid 66826)
Proper citation: RRID:Addgene_121062 Copy
Species: Caenorhabditis elegans
Genetic Insert: GFP-PH
Vector Backbone Description: Vector Backbone:pCFJ151; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:28811311
Proper citation: RRID:Addgene_107018 Copy
Species: Caenorhabditis elegans
Genetic Insert: mCherry(dpiRNA)::ANI-1(AHPH)
Vector Backbone Description: Vector Backbone:pCFJ150; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:29420292
Proper citation: RRID:Addgene_107939 Copy
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