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http://www.addgene.org/191023

Species: Caenorhabditis elegans
Genetic Insert: flp-18 CELE_Y48D7A.2
Vector Backbone Description: Vector Backbone:pPD95.62; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
References:
Comments:

Proper citation: RRID:Addgene_191023 Copy   

•   Source: Addgene

http://www.addgene.org/191020

Species: Caenorhabditis elegans
Genetic Insert: acr-5
Vector Backbone Description: Vector Backbone:pPD95.62; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
References:
Comments:

Proper citation: RRID:Addgene_191020 Copy   

•   Source: Addgene

http://www.addgene.org/191078

Species: Caenorhabditis elegans
Genetic Insert: flp-8
Vector Backbone Description: Vector Backbone:pPD95.62; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
References:
Comments:

Proper citation: RRID:Addgene_191078 Copy   

•   Source: Addgene

http://www.addgene.org/191073

Species: Caenorhabditis elegans
Genetic Insert: dop-6
Vector Backbone Description: Vector Backbone:pPD95.62; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
References:
Comments:

Proper citation: RRID:Addgene_191073 Copy   

•   Source: Addgene

http://www.addgene.org/191075

Species: Caenorhabditis elegans
Genetic Insert: flp-18(AVA = 4.2-1k)
Vector Backbone Description: Vector Backbone:pPD95.62; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
References:
Comments:

Proper citation: RRID:Addgene_191075 Copy   

•   Source: Addgene

http://www.addgene.org/191074

Species: Caenorhabditis elegans
Genetic Insert: flp-12
Vector Backbone Description: Vector Backbone:pPD95.62; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
References:
Comments:

Proper citation: RRID:Addgene_191074 Copy   

•   Source: Addgene

http://www.addgene.org/191534

Species: Caenorhabditis elegans
Genetic Insert: GtACR2
Vector Backbone Description: Backbone Marker:stratagene; Backbone Size:10495; Vector Backbone:pBSK; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
References:
Comments: Please visit for https://www.biorxiv.org/content/10.1101/2021.09.21.461278v4 bioRxiv preprint.

Proper citation: RRID:Addgene_191534 Copy   

•   Source: Addgene

http://www.addgene.org/191700

Species: Caenorhabditis elegans
Genetic Insert: GtACR2
Vector Backbone Description: Backbone Marker:stratagene; Backbone Size:7739; Vector Backbone:pBSK; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
References:
Comments: Please visit for https://www.biorxiv.org/content/10.1101/2021.09.21.461278v4 bioRxiv preprint.

Proper citation: RRID:Addgene_191700 Copy   

•   Source: Addgene

http://www.addgene.org/191702

Species: Caenorhabditis elegans
Genetic Insert: Tetanus Toxin Light Chain
Vector Backbone Description: Backbone Marker:stratagene; Backbone Size:9338; Vector Backbone:pBSK; Vector Types:Worm Expression; Bacterial Resistance:Ampicillin
References:
Comments: Please visit for https://www.biorxiv.org/content/10.1101/2021.09.21.461278v4 bioRxiv preprint.

Proper citation: RRID:Addgene_191702 Copy   

•   Source: Addgene

http://www.addgene.org/110930

Species: Caenorhabditis elegans
Genetic Insert: rab-3 promoter
Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
References:
Comments: Unique multi-cloning sites: Nhe I, Sbf I, Kpn I/ Age I/ Xho I, Bgl II This vector is the same as KG#59, except the Sal I and Acc I sites of the multi-cloning site have been replaced with a Sbf I site. Synthesized 2 complementary oligos containing Nhe I and Age I sticky ends and Sbf I and Kpn I sites in between. Hybridization of the 2 oligos to each other will produce Nhe I and Age I sticky ends. After hybridization, this synthetic insert (which is non-phosphorylated on its 5' termini) was cloned into Nhe I/ Age I cut KG#59. Transformed into XL1-Blue electrocompetent cells. Miniprepped 6 clones and tested for the Sbf I site by cutting with Sbf I. Chose one clone that has the Sbf I site with correct band size (and only a single band) and made the 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_110930 Copy   

•   Source: Addgene

http://www.addgene.org/110931

Species: Caenorhabditis elegans
Genetic Insert: unc-17 promoter
Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
References:
Comments: Unique multi-cloning sites: Nhe I, Sal I, Kpn I/ Age I/ Eco RV, Bgl II Used Apa I/ Msc I to cut out the ~1900 bp GFP/ 3' control region from RM#349p, leaving the 5900 bp vector fragment containing the unc-17 full promoter + remaining vector sequences. To this vector fragment, we ligated the 1079 bp Apa I/ Msc I fragment cut from pPD96.52 (C. elegans body wall muscle expression vector). Picked 6 colonies for minipreps. Chose 1 clone with correct size insert and made glycerol stock. Features of the expression construct: This is a C. elegans expression vector with the unc-17 3.2 Kb promoter + a large MCS for cloning in cDNAs. Includes cha-1/unc-17 upstream region from Sna-1 (=Bst1107 I = GTA/TAC) down to first half of the 66 bp first exon (which is common to unc-17 and cha-1 transcripts but is not translated). Includes beta site so that expression occurs in most cholinergic cells. Expression still missing from VC's and some cholinergic head and tail neurons. In this construct the GFP/ 3' control region of RM#349p is replaced with the MCS II + 3' control region of pPD96.52. This includes 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_110931 Copy   

•   Source: Addgene

http://www.addgene.org/110932

Species: Caenorhabditis elegans
Genetic Insert: unc-17 promoter
Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
References:
Comments: Unique multi-cloning sites: Nhe I, Sbf I, Kpn I/ Age I/ Eco RV, Bgl II Note: this vector is the same as KG#94, except a Sbf I site replaces the Sal I site. Synthesized 2 complementary oligos containing Nhe I and Age I sticky ends and Sbf I and Kpn I sites in between. Hybridization of the 2 oligos to each other should produce Nhe I and Age I sticky ends. After hybridization, this synthetic insert (which is non-phosphorylated on its 5' termini) was cloned into Nhe I/ Age I cut KG#94. Transformed into XL1-Blue electrocompetent cells. Miniprepped 4 clones and tested for the Sbf I site by cutting with Sbf I. Chose one clone that has the Sbf I site with correct band size (and only a single band) and made a glycerol stock. Features of the expression construct: This is a C. elegans expression vector with the unc-17 3.2 Kb promoter + a large MCS for cloning in cDNAs. Includes cha-1/unc-17 upstream region from Sna-1 (=Bst1107 I = GTA/TAC) down to first half of the 66 bp first exon (which is common to unc-17 and cha-1 transcripts but is not translated). Includes beta site so that expression occurs in most cholinergic cells. Expression still missing from VC's and some cholinergic head and tail neurons. In this construct the GFP/ 3' control region of RM#349p is replaced with the MCS II + 3' control region of pPD96.52. This includes 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_110932 Copy   

•   Source: Addgene

http://www.addgene.org/110882

Species: Caenorhabditis elegans
Genetic Insert: unc-129 promoter
Vector Backbone Description: Backbone Size:2610; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
References:
Comments: We used Herculase II polymerase and primers engineered with restriction sites to amplify the 1.2 Kb ctns-1 cDNA "a" isoform (minus its stop codon and in frame with downstream RFP) from KG#354 and cloned it into Nhe I/ Age I cut KG#240 (7.2 Kb; unc-129::____-mCherry). Transformed into XL1-Blue electrocompetent cells. Qiagen miniprepped 2 clones to find those with correct insert, then confirmed insert by sequencing. Reference: Edwards SL, Yu S, Hoover, CM, Phillips BC, Richmond JE, and Miller KG. (2013). An Organelle Gatekeeper Function for Caenorhabditis elegans UNC-16 (JIP3) at the Axon Initial Segment. Genetics. 194(1): 143-161.PMCID: PMC3632462. Features of the construct: ctns-1a is a marker for lysosomes. An AAAA precedes the start codon for a good consensus ribosome binding site. 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 DA/ DB cholinergic neurons of the ventral nerve cord. The 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). The stop codon is left off of the ctns-1 cDNA for in-frame fusion to the downstream RFP (mCherry). The Age I site encodes 2 amino acids that are in frame with the downstream RFP. There is an CAG following the restriction site that is not part of the following intron. The restriction site + these 3 nucleotides will code for the amino acids Thr, Gly, and Gln and those last 3 nucleotides (CAG) provide a good consensus for preceding an intron. After this is the 44 bp intron 3 from T07H6.4 followed by the mCherry cDNA sequence (711 bp; no further introns; this is the mCherry cDNA obtained directly from the Tsien lab at UCSD) and stop codon, then the unc-54 3' control region with the 2 introns (1 just upstream of the unc-54 sequence and 1 in the unc-54 sequence) in it because the Apa I site is the 3' boundary of all of this. This vector also includes the 5' UTR intron found in most Fire lab vectors. Another feature 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).

Proper citation: RRID:Addgene_110882 Copy   

•   Source: Addgene

http://www.addgene.org/110927

Species: Caenorhabditis elegans
Genetic Insert: unc-129 promoter
Vector Backbone Description: Backbone Size:2632; Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
References:
Comments: Unique multi-cloning sites: Nhe I/ Sal I/ Kpn I/ Age I/ Not I/ Bgl II We used Pst I/ Bam H I to cut out the ~1200 bp rab-3 promoter from KG#208, leaving the 3700 bp vector fragment. To this vector fragment, we ligated the 2600 bp Pst I/ Bam H I unc-129 promoter fragment cut from KG#230. Picked 4 colonies for minipreps. Chose 1 clone with correct size insert and made glycerol stock. Features of the expression construct: This is the same as KG#230 except it has a Not I site between the Xho I and Bgl II sites in the MC. 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_110927 Copy   

•   Source: Addgene

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
References:
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
References:
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
References:
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
References:
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
References:
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/110914

Species: Caenorhabditis elegans
Genetic Insert: rab-3 promoter
Vector Backbone Description: Vector Backbone:pUC; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
References:
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