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http://www.addgene.org/110925

Species: Caenorhabditis elegans
Genetic Insert: unc-129 promoter
Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:19797080
Comments: Unique multi-cloning sites: Nhe I/ Sal I/ Kpn I/ Age I/ Bgl II Used Pfu Ultra polymerase and primers engineered with restriction sites to amplify the 2.6 Kb unc-129 promoter from N2 genomic DNA and cloned it 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 4 clones to find those with correct size insert and vector, then submited 1 clone for sequence. Chose one clone that has correct sequence and made glycerol stock. Features of the expression construct: This expression construct has a promoter (unc-129::) that will drive strong and specific expression (of whatever gene is inserted in the MCS) in 9 of the DA/ DB cholinergic neurons of the ventral nerve cord. unc-129 promoter sequence includes the region from the native Sma I site (2.6 Kb upstream of the start codon) down to the 7th nucleotide before the A of the ATG. This includes a 19 bp 5' UTR (out of 25 bp 5' UTR total). This promoter is a shortened version of the unc-129 promoter. This shortened version drives expression in 9 ventral cord motor neurons that Sieburth et al identified as the DA's. The promoter itself is described in Colavita et al., 1998, although they claim expression in both DA's and DB's. The DA motor neurons form NMJ's in the dorsal nerve cord and receive synaptic inputs in the ventral cord (therefore proteins localized to the ventral cord would be dendritically localized/ postsynaptic). 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_110925 Copy   

•   Source: Addgene
  

http://www.addgene.org/110929

Species: Caenorhabditis elegans
Genetic Insert: rab-3 promoter
Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15489510
Comments: Unique multi-cloning sites: Nhe I, Sal I, Acc I, Kpn I/ Age I/ Xho I, Bgl II Used Pfu Ultra polymerase and primers engineered with restriction sites to amplify the 1.2 Kb rab-3 promoter from N2 genomic DNA and cloned it into Pst I/ Bam HI cut pPD96.52 (C. elegans body wall muscle expression vector; this digestion removed the myo-3 promoter in 2 fragments and left 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 glycerol stock. Features of the expression construct: This expression construct has a promoter (rab-3) that will drive strong and specific expression (of whatever gene is inserted in the MCS) in the entire nervous system of juvenile and adult animals. The boundaries of the rab-3 promoter sequence were obtained by examining pRabGFPrim3' from Mike Nonet's lab web site and comparing that to the genome sequence. 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_110929 Copy   

•   Source: Addgene
  

http://www.addgene.org/110876

Species: Caenorhabditis elegans
Genetic Insert: myo-3 promoter
Vector Backbone Description: Backbone Size:2617; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15489510
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(P260S) cDNA coding region from KG#63 and cloned into Age I/ Xho I cut pPD96.52 (myo-3:: expression vector). Transformed 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 make glycerol stock and save the DNA. Features of the expression construct: This expression construct has a promoter (myo-3) that will drive strong expression of acy-1 (P260S) cDNA in the body wall muscle cells. The myo-3 promoter is generally active in body muscles (body wall, vulval, and intestine associated muscles including the anal depressor [Okkema et al., 1993 Genetics; Ardizzi and Epstein 1987 JCB]. Other features of the expression vector include 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. 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 except for the gf mutation in this clone.

Proper citation: RRID:Addgene_110876 Copy   

•   Source: Addgene
  

http://www.addgene.org/110877

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:15489510
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 Age I/ Xho I to cut out the 3800 bp acy-1(P260S) cDNA from KG#81 and cloned into the like-digested rab-3 expression vector KG#59 (4900 bp). Transformed into XL1-Blue electrocompetent cells. Miniprepped 4 clones to find one with correct size insert and vector and made glycerol stock. Features of the construct: This expression construct has a promoter (rab-3) that will drive strong expression of the acy-1(P260S) cDNA in the nervous system. An unc-54 3' end containing a poly A addition signal is downstream of the MCS to provide a 3' UTR for the transcript. Three introns in the UTR's are included to help expression (1 in the 5' UTR, 1 just upstream of the unc-54 3' end, and 1 in the unc-54 3' end sequence). This construct includes only the acy-1 coding region. The sequence AAAA is engineered just after the 5' restriction site and just before the initiator ATG. An AT immediately follows the TAA stop codon. 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 except for the gf mutation in this clone.

Proper citation: RRID:Addgene_110877 Copy   

•   Source: Addgene
  

http://www.addgene.org/110878

Species: Caenorhabditis elegans
Genetic Insert: unc-17beta promoter
Vector Backbone Description: Backbone Size:2617; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15489510
Comments: Used Age I/ Xho I to cut out the 3800 bp acy-1(P260S) cDNA from KG#81 and cloned into the like-digested unc-17b expression vector KG#65 (4200 bp). Transformed into XL1-Blue electrocompetent cells. Miniprepped 4 clones to find one with correct size insert and vector and made glycerol stock. The insert was previously checked by sequencing and contains only the P260S mutation. Features of the construct: This expression construct has a promoter (unc-17beta) that will drive strong expression of the acy-1(P260S) cDNA in the A and B classes of ventral cord motor neurons + AS's, but not VC's or cholinergic neurons in the head or tail. The vector contains a "decoy" (see 1995 vector kit documentation) upstream of the promoter to reduce background expression in the gut and pharynx from read-through transcription. An unc-54 3' end containing a poly A addition signal is downstream of the MCS to stop transcription and provide a 3' UTR for the transcript. Three introns in the UTR's are included to give stable and uniform expression (1 in the 5' UTR, 1 just upstream of the unc-54 3' end, and 1 in the unc-54 3' end sequence). This construct includes only the coding region. The sequence AAAA is engineered just after the 5' restriction site and just before the initiator ATG. An AT immediately follows the TAA stop codon for a consensus stop site. 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_110878 Copy   

•   Source: Addgene
  

http://www.addgene.org/110911

Species: Caenorhabditis elegans
Genetic Insert: sup-1 minigene
Vector Backbone Description: Backbone Size:2124; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30254025
Comments: Made by IDT Custom Gene Synthesis. This sup-1 minigene can be inserted in C. elegans introns as a gene editing marker. The minigene begins with the 491 bp unc-17beta promoter. This is an engineered derivative of the full unc-17 promoter that drives expression specifically in cholinergic motor neurons of the ventral nerve cord (AS, DA, DB, VA, VB neuron classes) and not in cholinergic neurons elsewhere in the animal (Jim Rand, personal communication). The sup-1 portion of the minigene contains a single, small native intron (we removed a second, much larger intron) and includes the e995 suppressor mutation that rescues the unc-17(e245) mutant phenotype. The minigene finishes with the C. briggsae unc-119 3’ UTR, previously used in the unc-119 marker gene (FROKJAER-JENSEN et al. 2008). After searching the reverse orientation of the minigene sequence with the consensus spice acceptor sequence WTNYAG, we replaced 11 potential splice acceptors in the coding region, intron, and 3’ UTR with silent mutations. The AT content of the minigene is 63%, which approaches the 70% average for introns (BLUMENTHAL AND STEWARD 1997). The total size of the sup-1 minigene is 1112 bp.

Proper citation: RRID:Addgene_110911 Copy   

•   Source: Addgene
  

http://www.addgene.org/110914

Species: Caenorhabditis elegans
Genetic Insert: rab-3 promoter
Vector Backbone Description: Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Comments: We used Q5 polymerase and primers engineered with restriction sites to amplify the 1885 bp TIR1 sequence from pLZ31 and cloned it into Nhe I/ Kpn I cut KG#59 (4.9 Kb). Transformed into XL1-Blue electrocompetent cells. Did Qiagen preps on 2 clones to find one with correct size insert for sequence verification. Features of the construct: The rab-3:: promoter drives expression pan-neuronally. AAAA provides a consensus ribosome binding site. TAA AT provides a consensus stop translation site. This is the Arabidopsis thaliana (mustard weed) TIR1 sequence published in the Zhang et al., 2015 Auxin Inducible Degradation paper from the Dernburg lab. It has been codon optimized for C. elegans and contains 2 synthetic introns to boost expression. It also contains 2 point mutations (D170E and M473L) shown to increase the affinity of the Arabidopsis TIR1 protein for its substrates and to increase auxin sensitivity without causing auxin-independent activity. There are also introns in the 5' and 3' UTR in this vector.

Proper citation: RRID:Addgene_110914 Copy   

•   Source: Addgene
  

http://www.addgene.org/113062

Species: Caenorhabditis elegans
Genetic Insert: C113S-KVS-1
Vector Backbone Description: Vector Backbone:pCI-neo; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:19850480
Comments: This plasmid contains a K861I mutation in KVS-1 compared to the reference sequence AAO65852.1.

Proper citation: RRID:Addgene_113062 Copy   

•   Source: Addgene
  

http://www.addgene.org/113061

Species: Caenorhabditis elegans
Genetic Insert: K+ voltage sensitive channel subunit 1
Vector Backbone Description: Vector Backbone:pCI-neo; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16222231
Comments: This plasmid contains a K861I mutation in KVS-1 compared to the reference sequence AAO65852.1.

Proper citation: RRID:Addgene_113061 Copy   

•   Source: Addgene
  

http://www.addgene.org/113068

Species: Caenorhabditis elegans
Genetic Insert: C113S K+ voltage sensitive channel subunit 1.2
Vector Backbone Description: Vector Backbone:pCI-neo; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:19330004
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_113068 Copy   

•   Source: Addgene
  

http://www.addgene.org/113071

Species: Caenorhabditis elegans
Genetic Insert: MiRP K+ channel accessory Subunit 3
Vector Backbone Description: Vector Backbone:pCI-neo; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15799965
Comments: This plasmid contains a T61A mutation compared to the reference NP_490778.1 that does not impact function.

Proper citation: RRID:Addgene_113071 Copy   

•   Source: Addgene
  

http://www.addgene.org/113070

Species: Caenorhabditis elegans
Genetic Insert: C113S K+ voltage sensitive channel subunit 1
Vector Backbone Description: Vector Backbone:pCI-neo; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:19330004

Proper citation: RRID:Addgene_113070 Copy   

•   Source: Addgene
  

http://www.addgene.org/11337

Species: Caenorhabditis elegans
Genetic Insert: nud-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 F53A2.4, encoding the nud-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_11337 Copy   

•   Source: Addgene
  

http://www.addgene.org/11339

Species: Caenorhabditis elegans
Genetic Insert: nud-1
Vector Backbone Description: Backbone Marker:see pLexA map in "author's map"; Backbone Size:5700; Vector Backbone:pLexA; Vector Types:Yeast Expression; Bacterial Resistance:Ampicillin
Comments: Full-length cDNA for C. elegans open-reading frame F53A2.4, encoding the nud-1 gene, cloned into the yeast two-hybrid DNA-binding domain vector pLexA. This vector can be used as the bait for a yeast two-hybrid screen. See "author's map" for picture of empty pLexA vector. Please note that Addgene's sequencing result differs from GenBank ID NM_067348, which indicates mutations in nud-1 are present at K136R, C220G and E235G. 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_11339 Copy   

•   Source: Addgene
  

http://www.addgene.org/11338

Species: Caenorhabditis elegans
Genetic Insert: lis-1
Vector Backbone Description: Backbone Marker:see pLexA map in "author's map"; Backbone Size:5700; Vector Backbone:pLexA; Vector Types:Yeast Expression; Bacterial Resistance:Ampicillin
Comments: Full-length cDNA for C. elegans open-reading frame T03F6.5, encoding the lis-1 gene, cloned into the yeast two-hybrid DNA-binding domain vector pLexA. This vector can be used as the bait for a yeast two-hybrid screen. See "author's map" for picture of empty pLexA vector. 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_11338 Copy   

•   Source: Addgene
  

http://www.addgene.org/118223

Species: Caenorhabditis elegans
Genetic Insert: BioID (BirA mutant)
Vector Backbone Description: Backbone Size:6954; Vector Backbone:Bluescript; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30125270

Proper citation: RRID:Addgene_118223 Copy   

•   Source: Addgene
  

http://www.addgene.org/11906

Species: Caenorhabditis elegans
Genetic Insert: lin-41 miRNA target sequence
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 to test effects of lin-41 microRNA (let-7).

Proper citation: RRID:Addgene_11906 Copy   

•   Source: Addgene
  

http://www.addgene.org/121054

Species: Caenorhabditis elegans
Genetic Insert: GFP^SEC^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 pDD282 (AddGene plasmid 66823)

Proper citation: RRID:Addgene_121054 Copy   

•   Source: Addgene
  

http://www.addgene.org/121057

Species: Caenorhabditis elegans
Genetic Insert: mKate2-T^SEC^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_121057 Copy   

•   Source: Addgene
  

http://www.addgene.org/122253

Species: Caenorhabditis elegans
Genetic Insert: mNeptune
Vector Backbone Description: Vector Backbone:BSP188; Vector Types:Worm Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:33693300

Proper citation: RRID:Addgene_122253 Copy   

•   Source: Addgene