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Species: Synthetic
Genetic Insert: Full length TSWV S antigenome encoding sgRNA:GFP fusion in place of viral NSs
Vector Backbone Description: Vector Backbone:pCB301-2μ-HDV; Vector Types:Plant Expression, CRISPR; Bacterial Resistance:Kanamycin
Defining Citation: PMID:36751129
Proper citation: RRID:Addgene_196296 Copy
Species: Synthetic
Genetic Insert: AAV9 VP1 modified with 7mer insertion between amino acids 588 and 589
Vector Backbone Description: Backbone Size:3000; Vector Backbone:pUC57-Kan; Vector Types:AAV; Bacterial Resistance:Kanamycin
Defining Citation: PMID:36417917
Proper citation: RRID:Addgene_196691 Copy
Species: Homo sapiens
Genetic Insert: Codon optimized VPS13D
Vector Backbone Description: Backbone Marker:Genscript; Backbone Size:6000; Vector Backbone:pcDNA3.4; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:33891013
Proper citation: RRID:Addgene_194006 Copy
Species: Synthetic
Genetic Insert: proUBQ10::PIP2A-mCherry-NOSt_proG1090::AmtR-VP16-19St_1XAmtRO::GFPint-NLS-ADHt
Vector Backbone Description: Vector Backbone:JAB2155; Vector Types:Plant Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:35951698
Comments: Backbone JAB2155 contains proUBQ10::PIP2A-mCherry-NOSt_proG1090::AmtR-VP16-19St. Please see the fully annotated sequence linked the "Supplemental Documents" section. Please visit https://www.biorxiv.org/content/10.1101/2022.02.02.478917v1 for bioRxiv preprint.
Proper citation: RRID:Addgene_190386 Copy
Vector Backbone Description: Vector Backbone:pCB301; Vector Types:Plant Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:29112135
Proper citation: RRID:Addgene_196329 Copy
Species: Synthetic
Genetic Insert: 10His-pG-Tn5 (E54K, L372P)
Vector Backbone Description: Backbone Size:5234; Vector Backbone:pETv2; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:39026714
Comments: Please visit https://www.biorxiv.org/content/10.1101/2024.07.11.602973v1 for bioRxiv preprint.
Proper citation: RRID:Addgene_198467 Copy
Species: Synthetic
Genetic Insert: Full length TSWV L antigenome encoding codon-optimized L (RdRp)
Vector Backbone Description: Vector Backbone:pCB301-2μ-HDV; Vector Types:Plant Expression, CRISPR; Bacterial Resistance:Kanamycin
Defining Citation: PMID:36751129
Proper citation: RRID:Addgene_196286 Copy
Species: Synthetic
Genetic Insert: Full length TSWV M antigenome encoding CBE in place of viral GP
Vector Backbone Description: Vector Backbone:pCB301-2μ-HDV; Vector Types:Plant Expression, CRISPR; Bacterial Resistance:Kanamycin
Defining Citation: PMID:36751129
Proper citation: RRID:Addgene_196293 Copy
Species: Vibrio cholerae
Genetic Insert: VtrA-CadC fusion protein and cognate promoter inducible by secondary bile acids
Vector Backbone Description: Backbone Size:5601; Vector Backbone:pMUT2; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:36449712
Proper citation: RRID:Addgene_192862 Copy
Vector Backbone Description: Backbone Marker:Coleman; Backbone Size:4690; Vector Backbone:pUS250; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Kanamycin
Comments: CLONING: The multiple cloning site (MCS) in pUS250 functions differently to a typical MCS. You need to choose one unique restriction site on the left side of the amilCP marker gene (e.g. EcoRI), and one unique restriction site on the right side (e.g. PstI) in order for the blue/white selection and the cumate-inducible expression features to work properly. Cutting the plasmid in this way excises the amilCP gene, replacing it with your gene of interest, and changing the phenotype from blue to white. The vector is also compatible with GoldenGate cloning (using either BsaI or Esp3I) and BioBrick cloning (iGEM). In the case of GoldenGate cloning, you don't need to use two different enzymes, you just use one or the other, since each enzyme has two sites, one on each side of amilCP, and each yields a different overhang.
HOST RANGE and EXPRESSION: we have shown that pUS250 can be used for cumate-inducible gene expression in E.coli, Pseudomonas putida, and Rhizobium leguminosarum. It is likely that the plasmid will also be useful in other gram negatives (Proteobacteria) since the pBBR replicon has a very broad host range. The plasmid can be transferred by conjugation from E.coli strains such as S17 or SM10 into other species due to the oriT sequence. For the E.coli and Rhizobium, 100 uM cumate is sufficient for expression. For Pseudomonas, this needs to be increased to 10 mM (we think there is an efflux pump that pumps cumate back out of these cells). The cumate should be added after autoclaving. We make a 0.5 M cumate stock solution by mixing equal parts of 1M aqueous Tris base with 1M cumic acid in ethanol. Cumate is an excellent inducer since it is both cheap and non-toxic to both bacteria and people. You can even use cumin (the spice) for induction of gene expression in this plasmid! (there is enough cumate in the cumin).
STABILITY and COPY NUMBER: We estimate that the vector has a copy number of 5-10 in E.coli, so it is certainly a low copy vector. Best to make large-scale plasmid preps (e.g. 50 ml culture) rather than small-scale, in order to ensure you get enough plasmid DNA to work with. The plasmid is quite stable but we have seen white mutants appear occasionally which have deletions in amilCP.
Proper citation: RRID:Addgene_198322 Copy
Species: Bacterial
Genetic Insert: ER Halo(K73T)
Vector Backbone Description: Vector Backbone:pET30; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:35523806
Proper citation: RRID:Addgene_183692 Copy
Species: Mus musculus
Genetic Insert: mFirre 1.49 Clone
Vector Backbone Description: Backbone Size:4761; Vector Backbone:pDONR221; Vector Types:Gateway Cloning Intermediate; Bacterial Resistance:Kanamycin
Defining Citation: PMID:24463464
Proper citation: RRID:Addgene_195204 Copy
Species: Escherichia coli
Genetic Insert: lldR operon
Vector Backbone Description: Backbone Size:4866; Vector Backbone:pMUT2; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:36449712
Proper citation: RRID:Addgene_192859 Copy
Species: Escherichia coli
Genetic Insert: Acetoacetate inducible promoter
Vector Backbone Description: Backbone Size:5591; Vector Backbone:pMUT2; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:36449712
Proper citation: RRID:Addgene_192858 Copy
Species: Arabidopsis thaliana
Genetic Insert: tUBQ1
Vector Backbone Description: Backbone Marker:Thermo Fisher Scientific; Backbone Size:3512; Vector Backbone:pCR-BluntII-TOPO; Vector Types:Synthetic Biology; Bacterial Resistance:Kanamycin
Defining Citation: PMID:37012288
Proper citation: RRID:Addgene_195897 Copy
Species: Arabidopsis thaliana
Genetic Insert: tUBQ1
Vector Backbone Description: Backbone Marker:Thermo Fisher Scientific; Backbone Size:3512; Vector Backbone:pCR-BluntII-TOPO; Vector Types:Synthetic Biology; Bacterial Resistance:Kanamycin
Defining Citation: PMID:37012288
Proper citation: RRID:Addgene_195898 Copy
Species: Homo sapiens
Genetic Insert: FRB domain of mTOR
Vector Backbone Description: Vector Backbone:pEGFPN3; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_194120 Copy
Species: Synthetic
Genetic Insert: Intein-PhiC31
Vector Backbone Description: Backbone Marker:Thermo Fisher Scientific; Backbone Size:3512; Vector Backbone:pCR-BluntII-TOPO; Vector Types:Synthetic Biology; Bacterial Resistance:Kanamycin
Defining Citation: PMID:37012288
Proper citation: RRID:Addgene_195906 Copy
Vector Backbone Description: Backbone Size:5984; Vector Backbone:pGBT; Vector Types:Cre/Lox; Bacterial Resistance:Kanamycin
Defining Citation: PMID:32779569
Proper citation: RRID:Addgene_189848 Copy
Species: Burkholderia cenocepacia
Genetic Insert: IDH2 MTS-tl1 left TALE-G1397 DddA-C-UGI-SV40 polyA
Vector Backbone Description: Vector Backbone:pT3; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:34847747
Proper citation: RRID:Addgene_196506 Copy
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