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Species: Photorhabdus asymbiotica
Genetic Insert: PVCpnf_pvc13-A4DARPin
Vector Backbone Description: Vector Backbone:pAWP78; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:36991127
Comments: A446T mutation in pvc8 vgrG tail spike feature does not affect plasmid function.
Proper citation: RRID:Addgene_198288 Copy
Species: Synthetic
Genetic Insert: mCherry-CAAX
Vector Backbone Description: Backbone Size:7602; Vector Backbone:LV 1-5; Vector Types:Mammalian Expression, Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:34407185
Proper citation: RRID:Addgene_166228 Copy
Species: Photorhabdus asymbiotica
Genetic Insert: PVCpnf17-22
Vector Backbone Description: Vector Backbone:pBR322; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:36991127
Proper citation: RRID:Addgene_198272 Copy
Species: Other
Genetic Insert: mTagBFP2-CAAX
Vector Backbone Description: Backbone Size:8408; Vector Backbone:pLenti; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_197590 Copy
Species: Homo sapiens
Genetic Insert: GADD34 Δ1-240 - P2A - K3L
Vector Backbone Description: Backbone Size:7664; Vector Backbone:pSBtet-Hyg; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:36798401
Proper citation: RRID:Addgene_196136 Copy
Species: Mus musculus
Genetic Insert: bmal1
Vector Backbone Description: Vector Backbone:pCAGGS; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:35902736
Proper citation: RRID:Addgene_186829 Copy
Vector Backbone Description: Backbone Marker:TRC; Backbone Size:11079; Vector Backbone:PLKO.1; Vector Types:Mammalian Expression, Lentiviral, RNAi; Bacterial Resistance:Ampicillin
Defining Citation: PMID:39167658
Proper citation: RRID:Addgene_191567 Copy
Species: Homo sapiens
Genetic Insert: MEKDD
Vector Backbone Description: Vector Backbone:pLX311-GFP; Vector Types:Mammalian Expression, Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:36991492
Proper citation: RRID:Addgene_194882 Copy
Species: marine bacterium BMTU 3346 UDG
Genetic Insert: BMTU UDG
Vector Backbone Description: Backbone Marker:EMD Bioscience; Backbone Size:5443; Vector Backbone:pET-21(+); Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Comments: E. coli codon-optimised variant of BMTU 3346 UDG gene originally published in:
Jaeger, S., Schmuck, R., & Sobek, H. (2000). Molecular cloning, sequency, and expression of the heat-labile uracil-DNA glycosylase from a marine psychrophilic bacterium, strain BMTU3346. Extremophiles : life under extreme conditions, 4(2), 115–122. https://doi.org/10.1007/s007920050145
Proper citation: RRID:Addgene_172197 Copy
Species: Homo sapiens
Genetic Insert: NEUROG2 (phospho mutant, codon optimized)
Vector Backbone Description: Backbone Size:13243; Vector Backbone:pUCM; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_198397 Copy
Species: Synthetic
Genetic Insert: PEM7::tagrfp-T PtetA::sfgfp
Vector Backbone Description: Vector Backbone:pMW211; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:33947954
Comments: Please visit https://www.biorxiv.org/content/10.1101/2020.09.22.308114v1 for bioRxiv preprint.
Proper citation: RRID:Addgene_168177 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_196684 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_196687 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_196681 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: 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
Vector Backbone Description: Backbone Marker:Feng Zhang Lab; Vector Backbone:lentiGuide-Puro (#52963); Vector Types:Lentiviral, CRISPR; Bacterial Resistance:Ampicillin
Defining Citation: PMID:28993443
Proper citation: RRID:Addgene_196709 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: S. pyogenes
Genetic Insert: Cas9-T2A-BSD-U6-sgHPRT1
Vector Backbone Description: Backbone Marker:Feng Zhang Lab; Vector Backbone:lenti dCas9-VP64_Blast (#61425); Vector Types:Lentiviral, CRISPR; Bacterial Resistance:Ampicillin
Defining Citation: PMID:28993443
Proper citation: RRID:Addgene_196713 Copy
Species: S. pyogenes
Genetic Insert: dCas9-KRAB(ZIM3)-T2A-TagBFP
Vector Backbone Description: Backbone Marker:Feng Zhang Lab; Vector Backbone:lenti dCas9-VP64_Blast (#61425); Vector Types:Lentiviral, CRISPR; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_196712 Copy
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