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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/26387

Species: Caenorhabditis elegans
Vector Backbone Description: Backbone Size:0; Vector Backbone:pBS; Vector Types:Bacterial Expression, Worm Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20729841

Proper citation: RRID:Addgene_26387 Copy   

•   Source: Addgene

http://www.addgene.org/26347

Species: Caenorhabditis elegans
Genetic Insert: Pmyo-2::GFP::unc-54_3'UTR
Vector Backbone Description: Backbone Size:10813; Vector Backbone:pBCN27-R4R3; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20729840
Comments: The full sequence provided is theoretical sequence based on a three way gateway reaction. The order of the elements has been confirmed by PCR, but not fully sequence verified. Expression pattern in worms is as expected. Use this plasmid as a positive control for testing puromycin selection after single copy insertion by MosSCI at the ttTi5605 locus.

Proper citation: RRID:Addgene_26347 Copy   

•   Source: Addgene

http://www.addgene.org/26488

Species: Caenorhabditis elegans
Genetic Insert: cel-pre-let-7 hairpin only
Vector Backbone Description: Backbone Size:6946; Vector Backbone:MDH1-PGK-GFP; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20808284

Proper citation: RRID:Addgene_26488 Copy   

•   Source: Addgene

http://www.addgene.org/26491

Species: Caenorhabditis elegans
Genetic Insert: cel-let-7 (~250nt flanking each side)
Vector Backbone Description: Backbone Marker:Chang-Zheng Chen Lab; Backbone Size:7060; Vector Backbone:MXW-PGK-IRES-GFP; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20808284
Comments: Ordinarily, we express miRNAs from the LTR, which is pol III. However, cel-let-7 won't express from that promoter (possibly due to long poly-T tracts). This is a pol II expression system.

Proper citation: RRID:Addgene_26491 Copy   

•   Source: Addgene

http://www.addgene.org/26560

Species: Caenorhabditis elegans
Genetic Insert: cel-pre-let-7_LPB
Vector Backbone Description: Backbone Marker:Ambion; Backbone Size:2700; Vector Backbone:pDP19; Vector Types:transcription vector; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20808284
Comments: This construct does not produce perfect pre-let-7. There are leader and tail sequences from the backbone MCS. Used for in vitro translation.

Proper citation: RRID:Addgene_26560 Copy   

•   Source: Addgene

http://www.addgene.org/26559

Species: Caenorhabditis elegans
Genetic Insert: cel-pre-let-7_LPA
Vector Backbone Description: Backbone Marker:Ambion; Backbone Size:2700; Vector Backbone:pDP19; Vector Types:transcription vector; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20808284
Comments: This construct does not produce perfect pre-let-7. There are leader and tail sequences from the backbone MCS. Used for in vitro translation.

Proper citation: RRID:Addgene_26559 Copy   

•   Source: Addgene

http://www.addgene.org/26558

Species: Caenorhabditis elegans
Genetic Insert: cel-pre-let-7
Vector Backbone Description: Backbone Marker:Ambion; Backbone Size:2700; Vector Backbone:pDP19; Vector Types:transcription vector; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20808284
Comments: This construct does not produce perfect pre-let-7. There are leader and tail sequences from the backbone MCS. Loop mutants have also all been cloned into this backbone for in vitro translation.

Proper citation: RRID:Addgene_26558 Copy   

•   Source: Addgene

http://www.addgene.org/26938

Species: Caenorhabditis elegans
Genetic Insert: dcap-1
Vector Backbone Description: Backbone Size:0; Vector Backbone:unknown; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:18692039

Proper citation: RRID:Addgene_26938 Copy   

•   Source: Addgene

http://www.addgene.org/26814

Species: Caenorhabditis elegans
Genetic Insert: lag-2 promoter
Vector Backbone Description: Backbone Marker:Andrew Fire (Addgene Plasmid #1497); Backbone Size:4492; Vector Backbone:pPD95.81; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:7568229
Comments: Quick confirmation: BamHI should result in two fragments: ~3kb and 4.5kb. This is a lag-2 promoter::GFP fusion. Strong GFP in live worms. 3kb BamHI fragment of pJK520 is the complete 3kb 5' region of lag-2.

Proper citation: RRID:Addgene_26814 Copy   

•   Source: Addgene

http://www.addgene.org/21392

Species: Caenorhabditis elegans
Genetic Insert: cye-1 3'utr in pDONRP2RP3
Vector Backbone Description: Backbone Size:2641; Vector Backbone:pDONRP2RP3; Vector Types:Gateway Cloning; Bacterial Resistance:Kanamycin
Defining Citation: PMID:18818082

Proper citation: RRID:Addgene_21392 Copy   

•   Source: Addgene

http://www.addgene.org/21377

Species: Caenorhabditis elegans
Genetic Insert: L3787 promoter::GFP in pDONRP4P1R
Vector Backbone Description: Backbone Size:2645; Vector Backbone:pDONRP4P1R; Vector Types:Gateway Cloning; Bacterial Resistance:Kanamycin
Defining Citation: PMID:18818082

Proper citation: RRID:Addgene_21377 Copy   

•   Source: Addgene

http://www.addgene.org/21385

Species: Caenorhabditis elegans
Genetic Insert: spe-11 promoter (no ATG) in pDONRP4P1R
Vector Backbone Description: Backbone Size:2645; Vector Backbone:pDONRP4P1R; Vector Types:Gateway Cloning; Bacterial Resistance:Kanamycin
Defining Citation: PMID:18818082

Proper citation: RRID:Addgene_21385 Copy   

•   Source: Addgene

http://www.addgene.org/21384

Species: Caenorhabditis elegans
Genetic Insert: pie-1 promoter (short, no ATG) in pDONRP4P1R
Vector Backbone Description: Backbone Size:2645; Vector Backbone:pDONRP4P1R; Vector Types:Gateway Cloning; Bacterial Resistance:Kanamycin
Defining Citation: PMID:18818082

Proper citation: RRID:Addgene_21384 Copy   

•   Source: Addgene

http://www.addgene.org/21389

Species: Caenorhabditis elegans
Genetic Insert: gld-1 ORF+3' in pDONR201
Vector Backbone Description: Backbone Size:2257; Vector Backbone:pDONR201; Vector Types:Gateway Cloning; Bacterial Resistance:Kanamycin
Defining Citation: PMID:18818082

Proper citation: RRID:Addgene_21389 Copy   

•   Source: Addgene