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Species: Homo sapiens
Genetic Insert: Bcr
Vector Backbone Description: Backbone Size:7400; Vector Backbone:pLEF; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Anja Reichert. Generated in a 3-way ligation consisting of 5' 0.6 kb BamHI/SalI + 3' 3.5 kb SalI/XbaI BCR fragments into pLEF digested with BamHIxXbaI. The BCR insert contains the natural TAG and 3' UT sequences. At the 3' end there are polylinker sequences from pSK.
Proper citation: RRID:Addgene_38160 Copy
Species: Homo sapiens
Genetic Insert: Bcr/Abl P210
Vector Backbone Description: Backbone Marker:Pharmingen; Backbone Size:8530; Vector Backbone:pAcG2T; Vector Types:Insect Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Nora Heisterkamp. Made using a 3-way ligation with 0.6 kb 5' BamHI-SalI from BCR + 3' 7.5 kb Sal I-EcoRI into pAcG2T. Not evaluated for protein production.
Proper citation: RRID:Addgene_38161 Copy
Species: Homo sapiens
Genetic Insert: MITF-A
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4733; Vector Backbone:pEGFP-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
References:
Comments:
Proper citation: RRID:Addgene_38132 Copy
Species: Homo sapiens
Genetic Insert: cIAP1 H588A
Vector Backbone Description: Backbone Marker:Baltimore Lab; Backbone Size:5350; Vector Backbone:pEBB; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments:
Proper citation: RRID:Addgene_38233 Copy
Species: Homo sapiens
Genetic Insert: BCR
Vector Backbone Description: Backbone Size:4805; Vector Backbone:pKH3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Young Jin Cho.
A human BCR cDNA was inserted into pKH3 (Addgene plasmid #12555; obtained from Martin Schwartz) digested with BamHI x EcoRI in a three-way ligation (1.7 kb BamHI-SstI + 2.6 kb SstI-EcoRI). The 1.7 kb BamHI-SstI fragment was isolated from Bcr in pAcG2T (AddGene plasmid #38163). The 5’ sequence is
GGA TCC CCC GGG CTG CAG GAA TTC GAT GGC CGC GCC ATG-start BCR.
Proper citation: RRID:Addgene_38189 Copy
Species: Homo sapiens
Genetic Insert: BCR/ABL P190
Vector Backbone Description: Backbone Size:3000; Vector Backbone:pSK; Vector Types:construct for making transgenic mice; Bacterial Resistance:Ampicillin
References:
Comments: The DNA construct was made by Guido Jenster and Nora Heisterkamp in several cloning and subcloning steps. The transgenic construct contains human BCR exon 1, intron sequences of human BCR and ABL genes, human ABL exon 2 and ABL cDNA sequences. The transgene expression is controlled by a 200-bp Sst I- Bgl II fragment of the murine metallothionein-1 (Mt1) promoter lacking glucocorticoid-responsive elements (PMID 8541551). This BCR/ABL fusion gene produces a P190 protein and is a model for Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) in humans, which is caused by a chromosomal translocation that fuses the ABL proto-oncogene with breakpoint cluster region (BCR) sequences. Leukemia development is relatively rapid on inbred C57Bl/6J and CBA backgrounds and is slower on mixed genetic backgrounds. Mice transgenic for this construct on an inbred C57Bl/6J background are also available from Jackson labs stock 017833. The insert can be separated from the vector by digestion with Sst II.
Proper citation: RRID:Addgene_38185 Copy
Species: Homo sapiens
Genetic Insert: CRKL
Vector Backbone Description: Backbone Size:3500; Vector Backbone:pSL1180; Vector Types:mouse transgenic; Bacterial Resistance:Ampicillin
References:
Comments: This construct was made by Arnoud van Wijk. It can be used to make transgenic mice. The DNA construct contains 7.5 kb of human CRKL locus 5’ sequences including the promoter of the human v-crk sarcoma virus CT10 oncogene homolog (CRKL) gene, joined to CRKL exon 1. Part of the intron between exons 1 and 2 encompassing around 8-kb is included and exons 2-3 as cDNA. The entire insert is around 17 kb and can be isolated free from plasmid sequences in a Kpn I x Mlu I digestion. Jackson labs stock no. 017834 is one of our CRKL transgenics containing approximately 5 copies of this transgene. CRKL overexpressing transgenics are viable but mice with high copy numbers have reduced fertility.
How the CRKL transgenic construct was made: (verbatim from PMID 11245441): Human genomic clone 70 (PMID 7905853) contains 12 kb of 5’ sequences, exon 1, and 6.6 kb of intron 1 of the CRKL gene. A 350-bp RsaI fragment isolated from a CRKL cDNA (GenBank accession no. X59656) was used as a probe to specifically isolate a phage clone, CR-4, containing CRKL exon 2 flanked by 6.5 kb of intron 1 and 9.5 kb of intron 2. CRKL is located on human chromosome 22 and has been entirely sequenced; introns 1 and 2 are both around 15.5 kb. To generate a transgenic DNA construct, a 7-kb SalI-BamHI fragment from CR-4, which included a SalI site at the 5’ end from the phage polylinker and the 3’ BamHI site located in exon 2, was ligated with a 0.78-kb BamHI-EcoRI cDNA fragment, including exons 2, 3, and the 3’ untranslated region, into pSK digested with SalIxEcoRI. The insert was removed as a 7.7-kb SalI-NotI fragment and ligated with a 1.2-kb SstII-SalI fragment from intron 1 in clone 70 into pSK digested with SstII x NotI. The resulting insert was removed by digestion with SstII x NotI. The 5’ promoter and exon 1 sequences were isolated on an 8-kb EcoRI-SstII fragment that was subcloned into pSK digested with EcoRI x SstII; the insert was removed as an 8-kb KpnI-SstII fragment. The 8-kb KpnI-SstII fragment plus the 7.7-kb SstII-NotI fragment were ligated into pSL1180 digested with KpnI x NotI.
Proper citation: RRID:Addgene_38186 Copy
Species: Homo sapiens
Genetic Insert: autophagy-related genes 13
Vector Backbone Description: Backbone Marker:Dr. Toshio Kitamura of the University of Tokyo ; Backbone Size:5847; Vector Backbone:pMXs-IP; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
References:
Comments:
Proper citation: RRID:Addgene_38191 Copy
Species: Homo sapiens
Genetic Insert: ABR
Vector Backbone Description: Backbone Size:4805; Vector Backbone:pKH3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Young Jin Cho.
A human ABR cDNA was inserted into pKH3 (Addgene plasmid 12555; obtained from Martin Schwartz) digested with BamHI x EcoRI in a three-way ligation (0.4 kb BamHI-BstEII + 2.2 kb BstEII-EcoRI). The 5’ sequence is
GGA TCC ATG-start ABR. Note: This plasmid may contain an E24G mutation -gag to ggg- introduced by Taq polymerase at an earlier cloning step.
Proper citation: RRID:Addgene_38190 Copy
Species: Homo sapiens
Genetic Insert: Abr GAP domain
Vector Backbone Description: Backbone Size:5500; Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Young Jin Cho.
An 0.8 kb BamHI xEcoRI fragment from AbrGAP in pGEX3x (AddGene #36407) was inserted into pcDNA BamHI xEcoRI. The insert starts at a natural PvuII site in the ABR sequence. The fragment contains the natural TAG and 3’ UT sequences and encodes amino acid residues 597-859 (Abr isoform B).
Proper citation: RRID:Addgene_38177 Copy
Species: Homo sapiens
Genetic Insert: BCR
Vector Backbone Description: Backbone Size:6700; Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Young Jin Cho.
A 1.2 kb BamHI xEcoRI fragment from BcrGAP in pGEX3x (AddGene #36405) was inserted into pcDNA BamHI xEcoRI. Contains natural TAG and 3’ UT sequences. The construct includes amino acid residues 1004-1271.
Proper citation: RRID:Addgene_38175 Copy
Species: Homo sapiens
Genetic Insert: BCR GAP domain encoding residues 1004-1271 with R1090A point mutation
Vector Backbone Description: Backbone Size:5500; Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Young Jin Cho.
pcDNA3.1Xpress-BcrGAP plasmid(AddGene #38175) was digested by BstEII x EcoRI and a 1.2 kb fragment containing the R1090 wt site, TAG and 3’ UT sequences removed. The wt fragment was replaced by a 0.6 kb BstEII x EcoRI fragment containing the R1090A mutation isolated from EGFP-BCR R1090A pEGFP (AddGene #38173). The construct contains the natural TAG and 3’ UT sequences. The difference between wt and mutant plasmids is in the 3’ UT region. The construct includes amino acid residues 1004-1271.
Proper citation: RRID:Addgene_38176 Copy
Species: Homo sapiens
Genetic Insert: ABR DH domain N282AE283A
Vector Backbone Description: Backbone Size:5000; Vector Backbone:pGEX 3X; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Young Jin Cho.
Mutagenesis done using Stratagene Quick Change kit.
0.35 kb Bgl IIxSstI fragment containing the N282A/E283A double mutation from Abr EYFP (AddGene #36419) was ligated into the "ABR DH domain pGEX" plasmid (AddGene #38181) digested with Bgl II x SstI and removal of the 0.35 kb fragment containing the wt sequence N282E283. The amino acid numbering is as in NM_021962 (isoform A).
Proper citation: RRID:Addgene_38182 Copy
Species: Homo sapiens
Genetic Insert: FER
Vector Backbone Description: Backbone Marker:Pharmingen; Backbone Size:8530; Vector Backbone:pAcG2T; Vector Types:Insect Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Leena Haataja. 4-way ligation of BamHI/NsiI + NsiI/KpnI + KpnI/EcoRI fragments into pAcG2T digested with BamHI x EcoRI. The construct makes Fer protein but not from amplified virus stocks. Large-scale yield of protein is relatively low.
Proper citation: RRID:Addgene_38167 Copy
Species: Homo sapiens
Genetic Insert: ABR
Vector Backbone Description: Backbone Marker:Pharmingen; Backbone Size:8530; Vector Backbone:pAcG2T; Vector Types:Insect Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Leena Haataja. 4-way ligation of BamHI/StuI + StuI/BstEII + BstEII/EcoRI fragments into pAcG2T digested with BamHI x EcoRI. The construct makes Abr protein.
Addgene sequencing found an E24G point mutation--this does not alter plasmid function.
Proper citation: RRID:Addgene_38162 Copy
Species: Homo sapiens
Genetic Insert: BCR
Vector Backbone Description: Backbone Marker:Pharmingen; Backbone Size:8530; Vector Backbone:pAcG2T; Vector Types:Insect Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Vesa Kaartinen. Makes Bcr protein. Nucleotide sequence from BamHI cloning site to BCR ATG is:
5' GGA TCC CCC GGG CTG CAG GAA TTC GAT GGC CGC GCC ATG
Proper citation: RRID:Addgene_38163 Copy
Species: Homo sapiens
Genetic Insert: Rac1
Vector Backbone Description: Backbone Marker:Pharmingen; Backbone Size:8530; Vector Backbone:pAcG2T; Vector Types:Insect Expression; Bacterial Resistance:Ampicillin
References:
Comments: Constructed by Nora Heisterkamp. Rac1 cDNA isolated from K562 cDNA library. 5' BamHI site introduced by PCR. Constructed in 3-way ligation: 0.18 kb 5' BamHI-PvuII + 3' 0.4 kb PvuII-EcoRI into pAcG2T. Makes Rac1 in insect cells.
Proper citation: RRID:Addgene_38164 Copy
Species: Homo sapiens
Genetic Insert: ARRDC3
Vector Backbone Description: Backbone Marker:Invitrogen; Vector Backbone:pEF6; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments:
Proper citation: RRID:Addgene_38317 Copy
Species: Homo sapiens
Genetic Insert: TSG101
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5780; Vector Backbone:pEF6; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments: Please note that there is a mutation in the TSG101 ORF, Y82H. This mutation should not affect plasmid function.
Proper citation: RRID:Addgene_38318 Copy
Species: Homo sapiens
Genetic Insert: ARRDC1
Vector Backbone Description: Backbone Marker:Promega; Backbone Size:5472; Vector Backbone:pCI-neo; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
References:
Comments:
Proper citation: RRID:Addgene_38319 Copy
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