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Species: Mus musculus
Genetic Insert: Mus musculus GABA transporter 1
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: To fuse GFP to the C terminus of mGAT1, the mGAT1 open reading frame was subcloned into the HindIII and EcoRI sites of pcDNA3.1(+), and the GFP coding sequence was subcloned into the NotI and XbaI sites. A 12-residue spacer between mGAT1 and GFP was introduced by the multiple cloning site of the vector:
CGA ATT CTG CAG ATA TCC AGC ACA GTG GCG GCC GCC
R I L Q I S S T V A A A
Proper citation: RRID:Addgene_41662 Copy
Species: Mus musculus
Genetic Insert: Rad, GTP binding proteins (RGK)
Vector Backbone Description: Backbone Marker:Invitrogen; Vector Backbone:pcDNA4/His-Max C; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments:
Proper citation: RRID:Addgene_41654 Copy
Species: Mus musculus
Genetic Insert: Rem, GTP binding proteins (RGK)
Vector Backbone Description: Backbone Marker:Invitrogen; Vector Backbone:pcDNA4/His-Max C; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments:
Proper citation: RRID:Addgene_41651 Copy
Species: Mus musculus
Genetic Insert: SA-GFP reporter
Vector Backbone Description: Backbone Marker:Greg Donoho (Donoho, Jasin, & Berg, Mol Cell Biol. 1998) (PMID: 9632791); Backbone Size:2721; Vector Backbone:pGPD351 (modified from pBluescript); Vector Types:Mammalian Expression, Mouse Targeting, DSB repair reporter substrate; Bacterial Resistance:Ampicillin
References:
Comments: This construct encodes a novel chromosomal reporter substrate, SA-GFP, which that allow the introduction of a double-strand break (DSB) and which assays single-strand annealing (SSA). The DSB is generated by the rare-cutting endonuclease I-SceI, whose 18-bp recognition sequence has been integrated within the green fluorescent protein gene (GFP) in such a way that it disrupts the gene. The SA-GFP reporter consists of two tandem GFP gene fragments: 5'GFP and SceGFP3'. Repair of the I-SceI-generated DSB in SceGFP'3 by SSA results in a functional GFP gene when a DNA strand from SceGFP3' is annealed to the complementary strand of 5GFP', followed by appropriate DNA-processing steps. As a result, SSA between the homologous sequences in the GFP gene fragments produces a 2.7-kb deletion in the chromosome. The SA-GFP reporter can also be repaired by HDR, but this repair does not restore a functional GFP gene.
The hprtSAGFP construct has homology to the hypoxanthine phosphoribosyl transferase (hprt) locus and contains the selectable puromycin resistance gene (puroR) as well as the SSA reporter substrate SA-GFP. Cells that integrate this fragment at a random genomic location are resistant to puromycin, whereas cells in which the reporter is successfully targeted to the hprt locus are additionally resistant to 6-thioguanine (6-TG).
Green fluorescent protein (GFP) gene fragments for SA-GFP were derived from pEGFP-N1 (Clonetech). The SA-GFP reporter was cloned into the mouse hprt targeting vector pGPD351 (a kind gift of Greg Donoho; Donoho, Jasin, & Berg, Mol Cell Biol. 1998). The direction of transcription of the SceGFP gene is the same as that for hprt.
A complete annotated sequence is available for download in the supplementary information above.
Please Note: Addgene NGS unable to fully resolve CAG promoter sequence. Please see depositor sequence for that section of the plasmid.
Proper citation: RRID:Addgene_41594 Copy
Species: Mus musculus
Genetic Insert: Notch-1 Full Length
Vector Backbone Description: Backbone Size:4352; Vector Backbone:pCS2+MT; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: Alternate plasmid name: pCS2 NFL6MT
pCS+ NFL contains the Notch-1 protein up to amino acid 2184 fused to a hexameric Myc tag at the carboxy terminus.
Proper citation: RRID:Addgene_41728 Copy
Species: Mus musculus
Genetic Insert: Mus musculus GABA transporter 1
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: To generate the fluorescent mutants mGAT10XFP and mGAT1XFP* through mGAT1XFP45, the wild-type mGAT1 open reading frame (ORF) was subcloned without its original stop codon into the HindIII and EcoRI sites of the pcDNA3.1(+) expression vector multiple cloning site (MCS). XFP ORFs were then subcloned downstream from and in frame with the mGAT1 ORF at the NotI and XbaI sites of the pcDNA3.1(+) MCS. This resulted in a 12–amino acid spacer between the end of the mGAT1 sequence and the beginning of the fluorophore. The depositors modified a method for the integration of PCR fragments without the use of restriction enzymes (Geiser et al., 2001) to add the final 3, 8, 20, 28, or 45 codons of the human GAT1 (hGAT1) ORF. These were amplified from a source plasmid using the proof-reading PfuTurbo Cx Hotstart polymerase with 5′ and 3′ extensions corresponding to the 20–22-nt regions that flanked the intended site of insertion, such that the PCR product integrated in-frame immediately after the fluorophore sequence when used as the primers in a subsequent QuikChange II XL mutagenesis PCR reaction. For mGAT1XFP*, the depositors simply added a GTC codon for Val after the fluorophore ORF.
The attached image displays the protein sequences of the modified regions of mGAT1 for each fluorescent construct. mGAT10CFP and mGAT10YFP repeated the fusion design of mGAT10GFP but with the fluorophore exchanged as annotated. The three C-terminal residues of the mGAT0XFP fusions are -YKI-CO2−, which comprises a broadly defined consensus PDZ class II–interacting motif (X-φ-X-φ, where φ designates a hydrophobic residue and X any residue) (Sheng and Sala, 2001; Hung and Sheng, 2002). The depositors searched the Ensembl databases using Biomart (http://www.ebi.ac.uk/biomart) (Spudich et al., 2007) and applied the GO:0005886 “plasma membrane” cellular component filter. The search identified no known membrane proteins possessing the -YKI-CO2− C-terminal sequence.
In the mGAT1XFP* constructs, the depositors defined the terminal residue P(0) more narrowly, changing the terminal isoleucine residue present in mGAT10XFP to a valine in mGAT1XFP*. The resulting C-terminal sequence, -YKV-CO2−, reconstituted a functional PDZ class II–interacting motif present in Ephrin B receptors, a class that relies on interactions with the PDZ domain–containing proteins for clustering (Torres et al., 1998; Brückner et al., 1999; Lin et al., 1999; Madsen et al., 2005).
Other constructs in the C-terminal XFP fusion series, mGAT1XFP3, mGAT1XFP8, mGAT1XFP20, mGAT1XFP28, and mGAT1XFP45, had the most C-terminal 3, 8, 20, 28, or 45 residues of the hGAT1 appended after the mGAT1XFP fusion. The differences in nucleotide sequence between the hGAT1 and mGAT1 C termini were a useful source of positive identification when the depositors analyzed the clones during construction.
PCR integration was applied to amplify and insert EYFP or ECFP directly between residues R565 and L566, I570 and Q571, or V577 and R578 of mGAT1 to generate the mGAT15xxXFP5xxCT constructs. The site of XFP insertion in GAT1 is highlighted in the nomenclatures for these constructs by residue numbers flanking the fluorophore, and the “CT” denotes that the insertion occurs within the C terminus.
Please see the associated article for more detailed information regarding construct creation and usage.
Proper citation: RRID:Addgene_41679 Copy
Species: Mus musculus
Genetic Insert: Mus musculus GABA transporter 1
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: To generate the fluorescent mutants mGAT10XFP and mGAT1XFP* through mGAT1XFP45, the wild-type mGAT1 open reading frame (ORF) was subcloned without its original stop codon into the HindIII and EcoRI sites of the pcDNA3.1(+) expression vector multiple cloning site (MCS). XFP ORFs were then subcloned downstream from and in frame with the mGAT1 ORF at the NotI and XbaI sites of the pcDNA3.1(+) MCS. This resulted in a 12–amino acid spacer between the end of the mGAT1 sequence and the beginning of the fluorophore. The depositors modified a method for the integration of PCR fragments without the use of restriction enzymes (Geiser et al., 2001) to add the final 3, 8, 20, 28, or 45 codons of the human GAT1 (hGAT1) ORF. These were amplified from a source plasmid using the proof-reading PfuTurbo Cx Hotstart polymerase with 5′ and 3′ extensions corresponding to the 20–22-nt regions that flanked the intended site of insertion, such that the PCR product integrated in-frame immediately after the fluorophore sequence when used as the primers in a subsequent QuikChange II XL mutagenesis PCR reaction. For mGAT1XFP*, the depositors simply added a GTC codon for Val after the fluorophore ORF.
The attached image displays the protein sequences of the modified regions of mGAT1 for each fluorescent construct. mGAT10CFP and mGAT10YFP repeated the fusion design of mGAT10GFP but with the fluorophore exchanged as annotated. The three C-terminal residues of the mGAT0XFP fusions are -YKI-CO2−, which comprises a broadly defined consensus PDZ class II–interacting motif (X-φ-X-φ, where φ designates a hydrophobic residue and X any residue) (Sheng and Sala, 2001; Hung and Sheng, 2002). The depositors searched the Ensembl databases using Biomart (http://www.ebi.ac.uk/biomart) (Spudich et al., 2007) and applied the GO:0005886 “plasma membrane” cellular component filter. The search identified no known membrane proteins possessing the -YKI-CO2− C-terminal sequence.
In the mGAT1XFP* constructs, the depositors defined the terminal residue P(0) more narrowly, changing the terminal isoleucine residue present in mGAT10XFP to a valine in mGAT1XFP*. The resulting C-terminal sequence, -YKV-CO2−, reconstituted a functional PDZ class II–interacting motif present in Ephrin B receptors, a class that relies on interactions with the PDZ domain–containing proteins for clustering (Torres et al., 1998; Brückner et al., 1999; Lin et al., 1999; Madsen et al., 2005).
Other constructs in the C-terminal XFP fusion series, mGAT1XFP3, mGAT1XFP8, mGAT1XFP20, mGAT1XFP28, and mGAT1XFP45, had the most C-terminal 3, 8, 20, 28, or 45 residues of the hGAT1 appended after the mGAT1XFP fusion. The differences in nucleotide sequence between the hGAT1 and mGAT1 C termini were a useful source of positive identification when the depositors analyzed the clones during construction.
PCR integration was applied to amplify and insert EYFP or ECFP directly between residues R565 and L566, I570 and Q571, or V577 and R578 of mGAT1 to generate the mGAT15xxXFP5xxCT constructs. The site of XFP insertion in GAT1 is highlighted in the nomenclatures for these constructs by residue numbers flanking the fluorophore, and the “CT” denotes that the insertion occurs within the C terminus.
Please see the associated article for more detailed information regarding construct creation and usage.
Proper citation: RRID:Addgene_41677 Copy
Species: Mus musculus
Genetic Insert: Mus musculus GABA transporter 1
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: To generate the fluorescent mutants mGAT10XFP and mGAT1XFP* through mGAT1XFP45, the wild-type mGAT1 open reading frame (ORF) was subcloned without its original stop codon into the HindIII and EcoRI sites of the pcDNA3.1(+) expression vector multiple cloning site (MCS). XFP ORFs were then subcloned downstream from and in frame with the mGAT1 ORF at the NotI and XbaI sites of the pcDNA3.1(+) MCS. This resulted in a 12–amino acid spacer between the end of the mGAT1 sequence and the beginning of the fluorophore. The depositors modified a method for the integration of PCR fragments without the use of restriction enzymes (Geiser et al., 2001) to add the final 3, 8, 20, 28, or 45 codons of the human GAT1 (hGAT1) ORF. These were amplified from a source plasmid using the proof-reading PfuTurbo Cx Hotstart polymerase with 5′ and 3′ extensions corresponding to the 20–22-nt regions that flanked the intended site of insertion, such that the PCR product integrated in-frame immediately after the fluorophore sequence when used as the primers in a subsequent QuikChange II XL mutagenesis PCR reaction. For mGAT1XFP*, the depositors simply added a GTC codon for Val after the fluorophore ORF.
The attached image displays the protein sequences of the modified regions of mGAT1 for each fluorescent construct. mGAT10CFP and mGAT10YFP repeated the fusion design of mGAT10GFP but with the fluorophore exchanged as annotated. The three C-terminal residues of the mGAT0XFP fusions are -YKI-CO2−, which comprises a broadly defined consensus PDZ class II–interacting motif (X-φ-X-φ, where φ designates a hydrophobic residue and X any residue) (Sheng and Sala, 2001; Hung and Sheng, 2002). The depositors searched the Ensembl databases using Biomart (http://www.ebi.ac.uk/biomart) (Spudich et al., 2007) and applied the GO:0005886 “plasma membrane” cellular component filter. The search identified no known membrane proteins possessing the -YKI-CO2− C-terminal sequence.
In the mGAT1XFP* constructs, the depositors defined the terminal residue P(0) more narrowly, changing the terminal isoleucine residue present in mGAT10XFP to a valine in mGAT1XFP*. The resulting C-terminal sequence, -YKV-CO2−, reconstituted a functional PDZ class II–interacting motif present in Ephrin B receptors, a class that relies on interactions with the PDZ domain–containing proteins for clustering (Torres et al., 1998; Brückner et al., 1999; Lin et al., 1999; Madsen et al., 2005).
Other constructs in the C-terminal XFP fusion series, mGAT1XFP3, mGAT1XFP8, mGAT1XFP20, mGAT1XFP28, and mGAT1XFP45, had the most C-terminal 3, 8, 20, 28, or 45 residues of the hGAT1 appended after the mGAT1XFP fusion. The differences in nucleotide sequence between the hGAT1 and mGAT1 C termini were a useful source of positive identification when the depositors analyzed the clones during construction.
PCR integration was applied to amplify and insert EYFP or ECFP directly between residues R565 and L566, I570 and Q571, or V577 and R578 of mGAT1 to generate the mGAT15xxXFP5xxCT constructs. The site of XFP insertion in GAT1 is highlighted in the nomenclatures for these constructs by residue numbers flanking the fluorophore, and the “CT” denotes that the insertion occurs within the C terminus.
Please see the associated article for more detailed information regarding construct creation and usage.
Proper citation: RRID:Addgene_41673 Copy
Species: Mus musculus
Genetic Insert: Mus musculus GABA transporter 1
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: To generate the fluorescent mutants mGAT10XFP and mGAT1XFP* through mGAT1XFP45, the wild-type mGAT1 open reading frame (ORF) was subcloned without its original stop codon into the HindIII and EcoRI sites of the pcDNA3.1(+) expression vector multiple cloning site (MCS). XFP ORFs were then subcloned downstream from and in frame with the mGAT1 ORF at the NotI and XbaI sites of the pcDNA3.1(+) MCS. This resulted in a 12–amino acid spacer between the end of the mGAT1 sequence and the beginning of the fluorophore. The depositors modified a method for the integration of PCR fragments without the use of restriction enzymes (Geiser et al., 2001) to add the final 3, 8, 20, 28, or 45 codons of the human GAT1 (hGAT1) ORF. These were amplified from a source plasmid using the proof-reading PfuTurbo Cx Hotstart polymerase with 5′ and 3′ extensions corresponding to the 20–22-nt regions that flanked the intended site of insertion, such that the PCR product integrated in-frame immediately after the fluorophore sequence when used as the primers in a subsequent QuikChange II XL mutagenesis PCR reaction. For mGAT1XFP*, the depositors simply added a GTC codon for Val after the fluorophore ORF.
The attached image displays the protein sequences of the modified regions of mGAT1 for each fluorescent construct. mGAT10CFP and mGAT10YFP repeated the fusion design of mGAT10GFP but with the fluorophore exchanged as annotated. The three C-terminal residues of the mGAT0XFP fusions are -YKI-CO2−, which comprises a broadly defined consensus PDZ class II–interacting motif (X-φ-X-φ, where φ designates a hydrophobic residue and X any residue) (Sheng and Sala, 2001; Hung and Sheng, 2002). The depositors searched the Ensembl databases using Biomart (http://www.ebi.ac.uk/biomart) (Spudich et al., 2007) and applied the GO:0005886 “plasma membrane” cellular component filter. The search identified no known membrane proteins possessing the -YKI-CO2− C-terminal sequence.
In the mGAT1XFP* constructs, the depositors defined the terminal residue P(0) more narrowly, changing the terminal isoleucine residue present in mGAT10XFP to a valine in mGAT1XFP*. The resulting C-terminal sequence, -YKV-CO2−, reconstituted a functional PDZ class II–interacting motif present in Ephrin B receptors, a class that relies on interactions with the PDZ domain–containing proteins for clustering (Torres et al., 1998; Brückner et al., 1999; Lin et al., 1999; Madsen et al., 2005).
Other constructs in the C-terminal XFP fusion series, mGAT1XFP3, mGAT1XFP8, mGAT1XFP20, mGAT1XFP28, and mGAT1XFP45, had the most C-terminal 3, 8, 20, 28, or 45 residues of the hGAT1 appended after the mGAT1XFP fusion. The differences in nucleotide sequence between the hGAT1 and mGAT1 C termini were a useful source of positive identification when the depositors analyzed the clones during construction.
PCR integration was applied to amplify and insert EYFP or ECFP directly between residues R565 and L566, I570 and Q571, or V577 and R578 of mGAT1 to generate the mGAT15xxXFP5xxCT constructs. The site of XFP insertion in GAT1 is highlighted in the nomenclatures for these constructs by residue numbers flanking the fluorophore, and the “CT” denotes that the insertion occurs within the C terminus.
Please see the associated article for more detailed information regarding construct creation and usage.
Proper citation: RRID:Addgene_41671 Copy
Species: Mus musculus
Genetic Insert: Mus musculus GABA transporter 1
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: To generate the fluorescent mutants mGAT10XFP and mGAT1XFP* through mGAT1XFP45, the wild-type mGAT1 open reading frame (ORF) was subcloned without its original stop codon into the HindIII and EcoRI sites of the pcDNA3.1(+) expression vector multiple cloning site (MCS). XFP ORFs were then subcloned downstream from and in frame with the mGAT1 ORF at the NotI and XbaI sites of the pcDNA3.1(+) MCS. This resulted in a 12–amino acid spacer between the end of the mGAT1 sequence and the beginning of the fluorophore. The depositors modified a method for the integration of PCR fragments without the use of restriction enzymes (Geiser et al., 2001) to add the final 3, 8, 20, 28, or 45 codons of the human GAT1 (hGAT1) ORF. These were amplified from a source plasmid using the proof-reading PfuTurbo Cx Hotstart polymerase with 5′ and 3′ extensions corresponding to the 20–22-nt regions that flanked the intended site of insertion, such that the PCR product integrated in-frame immediately after the fluorophore sequence when used as the primers in a subsequent QuikChange II XL mutagenesis PCR reaction. For mGAT1XFP*, the depositors simply added a GTC codon for Val after the fluorophore ORF.
The attached image displays the protein sequences of the modified regions of mGAT1 for each fluorescent construct. mGAT10CFP and mGAT10YFP repeated the fusion design of mGAT10GFP but with the fluorophore exchanged as annotated. The three C-terminal residues of the mGAT0XFP fusions are -YKI-CO2−, which comprises a broadly defined consensus PDZ class II–interacting motif (X-φ-X-φ, where φ designates a hydrophobic residue and X any residue) (Sheng and Sala, 2001; Hung and Sheng, 2002). The depositors searched the Ensembl databases using Biomart (http://www.ebi.ac.uk/biomart) (Spudich et al., 2007) and applied the GO:0005886 “plasma membrane” cellular component filter. The search identified no known membrane proteins possessing the -YKI-CO2− C-terminal sequence.
In the mGAT1XFP* constructs, the depositors defined the terminal residue P(0) more narrowly, changing the terminal isoleucine residue present in mGAT10XFP to a valine in mGAT1XFP*. The resulting C-terminal sequence, -YKV-CO2−, reconstituted a functional PDZ class II–interacting motif present in Ephrin B receptors, a class that relies on interactions with the PDZ domain–containing proteins for clustering (Torres et al., 1998; Brückner et al., 1999; Lin et al., 1999; Madsen et al., 2005).
Other constructs in the C-terminal XFP fusion series, mGAT1XFP3, mGAT1XFP8, mGAT1XFP20, mGAT1XFP28, and mGAT1XFP45, had the most C-terminal 3, 8, 20, 28, or 45 residues of the hGAT1 appended after the mGAT1XFP fusion. The differences in nucleotide sequence between the hGAT1 and mGAT1 C termini were a useful source of positive identification when the depositors analyzed the clones during construction.
PCR integration was applied to amplify and insert EYFP or ECFP directly between residues R565 and L566, I570 and Q571, or V577 and R578 of mGAT1 to generate the mGAT15xxXFP5xxCT constructs. The site of XFP insertion in GAT1 is highlighted in the nomenclatures for these constructs by residue numbers flanking the fluorophore, and the “CT” denotes that the insertion occurs within the C terminus.
Please see the associated article for more detailed information regarding construct creation and usage.
Proper citation: RRID:Addgene_41670 Copy
Species: Mus musculus
Genetic Insert: Mus musculus GABA transporter 1
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: To generate the fluorescent mutants mGAT10XFP and mGAT1XFP* through mGAT1XFP45, the wild-type mGAT1 open reading frame (ORF) was subcloned without its original stop codon into the HindIII and EcoRI sites of the pcDNA3.1(+) expression vector multiple cloning site (MCS). XFP ORFs were then subcloned downstream from and in frame with the mGAT1 ORF at the NotI and XbaI sites of the pcDNA3.1(+) MCS. This resulted in a 12–amino acid spacer between the end of the mGAT1 sequence and the beginning of the fluorophore. The depositors modified a method for the integration of PCR fragments without the use of restriction enzymes (Geiser et al., 2001) to add the final 3, 8, 20, 28, or 45 codons of the human GAT1 (hGAT1) ORF. These were amplified from a source plasmid using the proof-reading PfuTurbo Cx Hotstart polymerase with 5′ and 3′ extensions corresponding to the 20–22-nt regions that flanked the intended site of insertion, such that the PCR product integrated in-frame immediately after the fluorophore sequence when used as the primers in a subsequent QuikChange II XL mutagenesis PCR reaction. For mGAT1XFP*, the depositors simply added a GTC codon for Val after the fluorophore ORF.
The attached image displays the protein sequences of the modified regions of mGAT1 for each fluorescent construct. mGAT10CFP and mGAT10YFP repeated the fusion design of mGAT10GFP but with the fluorophore exchanged as annotated. The three C-terminal residues of the mGAT0XFP fusions are -YKI-CO2−, which comprises a broadly defined consensus PDZ class II–interacting motif (X-φ-X-φ, where φ designates a hydrophobic residue and X any residue) (Sheng and Sala, 2001; Hung and Sheng, 2002). The depositors searched the Ensembl databases using Biomart (http://www.ebi.ac.uk/biomart) (Spudich et al., 2007) and applied the GO:0005886 “plasma membrane” cellular component filter. The search identified no known membrane proteins possessing the -YKI-CO2− C-terminal sequence.
In the mGAT1XFP* constructs, the depositors defined the terminal residue P(0) more narrowly, changing the terminal isoleucine residue present in mGAT10XFP to a valine in mGAT1XFP*. The resulting C-terminal sequence, -YKV-CO2−, reconstituted a functional PDZ class II–interacting motif present in Ephrin B receptors, a class that relies on interactions with the PDZ domain–containing proteins for clustering (Torres et al., 1998; Brückner et al., 1999; Lin et al., 1999; Madsen et al., 2005).
Other constructs in the C-terminal XFP fusion series, mGAT1XFP3, mGAT1XFP8, mGAT1XFP20, mGAT1XFP28, and mGAT1XFP45, had the most C-terminal 3, 8, 20, 28, or 45 residues of the hGAT1 appended after the mGAT1XFP fusion. The differences in nucleotide sequence between the hGAT1 and mGAT1 C termini were a useful source of positive identification when the depositors analyzed the clones during construction.
PCR integration was applied to amplify and insert EYFP or ECFP directly between residues R565 and L566, I570 and Q571, or V577 and R578 of mGAT1 to generate the mGAT15xxXFP5xxCT constructs. The site of XFP insertion in GAT1 is highlighted in the nomenclatures for these constructs by residue numbers flanking the fluorophore, and the “CT” denotes that the insertion occurs within the C terminus.
Please see the associated article for more detailed information regarding construct creation and usage.
Proper citation: RRID:Addgene_41669 Copy
Species: Mus musculus
Genetic Insert: Mus musculus GABA transporter 1
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: To generate the fluorescent mutants mGAT10XFP and mGAT1XFP* through mGAT1XFP45, the wild-type mGAT1 open reading frame (ORF) was subcloned without its original stop codon into the HindIII and EcoRI sites of the pcDNA3.1(+) expression vector multiple cloning site (MCS). XFP ORFs were then subcloned downstream from and in frame with the mGAT1 ORF at the NotI and XbaI sites of the pcDNA3.1(+) MCS. This resulted in a 12–amino acid spacer between the end of the mGAT1 sequence and the beginning of the fluorophore. The depositors modified a method for the integration of PCR fragments without the use of restriction enzymes (Geiser et al., 2001) to add the final 3, 8, 20, 28, or 45 codons of the human GAT1 (hGAT1) ORF. These were amplified from a source plasmid using the proof-reading PfuTurbo Cx Hotstart polymerase with 5′ and 3′ extensions corresponding to the 20–22-nt regions that flanked the intended site of insertion, such that the PCR product integrated in-frame immediately after the fluorophore sequence when used as the primers in a subsequent QuikChange II XL mutagenesis PCR reaction. For mGAT1XFP*, the depositors simply added a GTC codon for Val after the fluorophore ORF.
The attached image displays the protein sequences of the modified regions of mGAT1 for each fluorescent construct. mGAT10CFP and mGAT10YFP repeated the fusion design of mGAT10GFP but with the fluorophore exchanged as annotated. The three C-terminal residues of the mGAT0XFP fusions are -YKI-CO2−, which comprises a broadly defined consensus PDZ class II–interacting motif (X-φ-X-φ, where φ designates a hydrophobic residue and X any residue) (Sheng and Sala, 2001; Hung and Sheng, 2002). The depositors searched the Ensembl databases using Biomart (http://www.ebi.ac.uk/biomart) (Spudich et al., 2007) and applied the GO:0005886 “plasma membrane” cellular component filter. The search identified no known membrane proteins possessing the -YKI-CO2− C-terminal sequence.
In the mGAT1XFP* constructs, the depositors defined the terminal residue P(0) more narrowly, changing the terminal isoleucine residue present in mGAT10XFP to a valine in mGAT1XFP*. The resulting C-terminal sequence, -YKV-CO2−, reconstituted a functional PDZ class II–interacting motif present in Ephrin B receptors, a class that relies on interactions with the PDZ domain–containing proteins for clustering (Torres et al., 1998; Brückner et al., 1999; Lin et al., 1999; Madsen et al., 2005).
Other constructs in the C-terminal XFP fusion series, mGAT1XFP3, mGAT1XFP8, mGAT1XFP20, mGAT1XFP28, and mGAT1XFP45, had the most C-terminal 3, 8, 20, 28, or 45 residues of the hGAT1 appended after the mGAT1XFP fusion. The differences in nucleotide sequence between the hGAT1 and mGAT1 C termini were a useful source of positive identification when the depositors analyzed the clones during construction.
PCR integration was applied to amplify and insert EYFP or ECFP directly between residues R565 and L566, I570 and Q571, or V577 and R578 of mGAT1 to generate the mGAT15xxXFP5xxCT constructs. The site of XFP insertion in GAT1 is highlighted in the nomenclatures for these constructs by residue numbers flanking the fluorophore, and the “CT” denotes that the insertion occurs within the C terminus.
Please see the associated article for more detailed information regarding construct creation and usage.
Proper citation: RRID:Addgene_41666 Copy
Species: Mus musculus
Genetic Insert: Mus musculus GABA transporter 1
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: To generate the fluorescent mutants mGAT10XFP and mGAT1XFP* through mGAT1XFP45, the wild-type mGAT1 open reading frame (ORF) was subcloned without its original stop codon into the HindIII and EcoRI sites of the pcDNA3.1(+) expression vector multiple cloning site (MCS). XFP ORFs were then subcloned downstream from and in frame with the mGAT1 ORF at the NotI and XbaI sites of the pcDNA3.1(+) MCS. This resulted in a 12–amino acid spacer between the end of the mGAT1 sequence and the beginning of the fluorophore. The depositors modified a method for the integration of PCR fragments without the use of restriction enzymes (Geiser et al., 2001) to add the final 3, 8, 20, 28, or 45 codons of the human GAT1 (hGAT1) ORF. These were amplified from a source plasmid using the proof-reading PfuTurbo Cx Hotstart polymerase with 5′ and 3′ extensions corresponding to the 20–22-nt regions that flanked the intended site of insertion, such that the PCR product integrated in-frame immediately after the fluorophore sequence when used as the primers in a subsequent QuikChange II XL mutagenesis PCR reaction. For mGAT1XFP*, the depositors simply added a GTC codon for Val after the fluorophore ORF.
The attached image displays the protein sequences of the modified regions of mGAT1 for each fluorescent construct. mGAT10CFP and mGAT10YFP repeated the fusion design of mGAT10GFP but with the fluorophore exchanged as annotated. The three C-terminal residues of the mGAT0XFP fusions are -YKI-CO2−, which comprises a broadly defined consensus PDZ class II–interacting motif (X-φ-X-φ, where φ designates a hydrophobic residue and X any residue) (Sheng and Sala, 2001; Hung and Sheng, 2002). The depositors searched the Ensembl databases using Biomart (http://www.ebi.ac.uk/biomart) (Spudich et al., 2007) and applied the GO:0005886 “plasma membrane” cellular component filter. The search identified no known membrane proteins possessing the -YKI-CO2− C-terminal sequence.
In the mGAT1XFP* constructs, the depositors defined the terminal residue P(0) more narrowly, changing the terminal isoleucine residue present in mGAT10XFP to a valine in mGAT1XFP*. The resulting C-terminal sequence, -YKV-CO2−, reconstituted a functional PDZ class II–interacting motif present in Ephrin B receptors, a class that relies on interactions with the PDZ domain–containing proteins for clustering (Torres et al., 1998; Brückner et al., 1999; Lin et al., 1999; Madsen et al., 2005).
Other constructs in the C-terminal XFP fusion series, mGAT1XFP3, mGAT1XFP8, mGAT1XFP20, mGAT1XFP28, and mGAT1XFP45, had the most C-terminal 3, 8, 20, 28, or 45 residues of the hGAT1 appended after the mGAT1XFP fusion. The differences in nucleotide sequence between the hGAT1 and mGAT1 C termini were a useful source of positive identification when the depositors analyzed the clones during construction.
PCR integration was applied to amplify and insert EYFP or ECFP directly between residues R565 and L566, I570 and Q571, or V577 and R578 of mGAT1 to generate the mGAT15xxXFP5xxCT constructs. The site of XFP insertion in GAT1 is highlighted in the nomenclatures for these constructs by residue numbers flanking the fluorophore, and the “CT” denotes that the insertion occurs within the C terminus.
Please see the associated article for more detailed information regarding construct creation and usage.
Proper citation: RRID:Addgene_41665 Copy
Species: Mus musculus
Genetic Insert: Klf5
Vector Backbone Description: Backbone Marker:Clontech; Vector Backbone:pRev-Tre; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments:
Proper citation: RRID:Addgene_41744 Copy
Species: Mus musculus
Genetic Insert: Notch-1 Lin-Notch-glp (LNG) repeat-containing construct
Vector Backbone Description: Backbone Size:4352; Vector Backbone:pCS2+MT; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: Alternate plasmid name: pCS2 NLNR-6MT
All Notch1 constructs deposited here were cloned into the pCS2+MT vector (Rupp et al., 1994; Turner and Weintraub, 1994) and have the C-terminal 348 residues (aa 2185 to C terminus) of Notch replaced with a hexameric myc tag to facilitate biochemical analysis.
Proper citation: RRID:Addgene_41738 Copy
Species: Mus musculus
Genetic Insert: Notch-1 Extracellular-domain-deleted construct
Vector Backbone Description: Backbone Size:4352; Vector Backbone:pCS2+MT; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: Alternate plasmid name: pCS2 NΔE-6MT
All Notch1 constructs deposited here were cloned into the pCS2+MT vector (Rupp et al., 1994; Turner and Weintraub, 1994) and have the C-terminal 348 residues (aa 2185 to C terminus) of Notch replaced with a hexameric myc tag to facilitate biochemical analysis.
Proper citation: RRID:Addgene_41737 Copy
Species: Mus musculus
Genetic Insert: p67 myb binding protein
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5500; Vector Backbone:pcDNA3.1 myc-His A; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments:
Proper citation: RRID:Addgene_42 Copy
Species: Mus musculus
Genetic Insert: Trp 53
Vector Backbone Description: Backbone Size:10180; Vector Backbone:pCXLE; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments:
Proper citation: RRID:Addgene_41859 Copy
Species: Mus musculus
Genetic Insert: Frizzled 1
Vector Backbone Description: Vector Backbone:pRK5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments:
Proper citation: RRID:Addgene_42253 Copy
Species: Mus musculus
Genetic Insert: Frizzled 3
Vector Backbone Description: Vector Backbone:pRK5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments:
Proper citation: RRID:Addgene_42255 Copy
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