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Species: HIV-1
Genetic Insert: HIV-1 Envelope Glycoprotein
Vector Backbone Description: Backbone Marker:Invitrogen; Vector Backbone: pcDNA3.1D/V5-His-TOPO; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:29020646
Comments: HIV-1 infected Patient Samples from Singapore, reverse transcribed RNA cloned into expression vector
Proper citation: RRID:Addgene_137691 Copy
Vector Backbone Description: Backbone Marker:Feng Zhang Lab; Backbone Size:10963; Vector Backbone:lentiGuide-Puro-P2A-EGFP; Vector Types:Mammalian Expression, Bacterial Expression, Mouse Targeting, Lentiviral, CRISPR, Synthetic Biology; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31980518
Comments: LentiGuide-Puro-P2A-GFP_mRFPstuf was generated from the lentiGuide-Puro-P2A-GFP (#137729), which was generated from lentiGuide-Puro (#52963).
The plasmid has mRFP driven by the bacterial lac promotor in the stuffer (the mRFP cassette was a kind gift from Dr. Erik Holmgren). The stuffer, 2839 bp, is excised by BsmBI digestion and replaced by a CRISPR library (or individual gRNA spacers). If the BsmBI digestion and the subsequent cloning is inadequate, the resulting CRISPR library can be contaminated by plasmids containing the stuffer instead of gRNAs. The bacterial mRFP cassette in the stuffer enables rapid identification of bacterial colonies carrying plasmids with retained stuffer (bacteria are red) and colonies without the stuffer (bacteria are white). This can be used as a simple visual quality control step for the gRNA cloning efficiency, as well as guide the selection of bacterial colonies where the stuffer has been excised.
A unique EcoRI site was introduced directly following the sgRNA scaffold, facilitating cloning in this region (introducing UMIs, introducing mutations in the sgRNA scaffold, etc).
The plasmid contains both PuroR and EGFP, enabling the selection of successfully infected cells based on both these selection markers. We typically add a pre-determined dose of puromycin (~5-15ug/ml) 24h after infection, using non-infected cells as controls for the efficiency of the selection. After 24-48h selection all non-infected control cells are typically dead, and the infected cells are ~100% EGFP positive. The EGFP signal can also be used for the simple determination of MOI of the viral preparation.
The provided sequencing primers are the primers traditionally used for NGS sequencing of gRNA representation in lentiGuide-Puro (#52963). This primer pair can also be used for Sanger sequencing the insert, amplifying the DNA sample (60oC annealing temp), and sequencing the ~300bp amplicon using the reversed primer. The forward primer is too close to the gRNA sequence for Sanger sequence. 5’-TGTAAACACAAAGATATTAGTACAAAATACGTGAC-3´ can be used as an alternative forward primer, resulting in a larger amplicon, better suited for Sanger sequencing.
LentiGuide-Puro-P2A-GFP_mRFPstuf vector was generated from the lentiGuide-Puro-P2A-GFP vector (#137729). This construct has RFP driven by a bacterial promotor in the stuffer (a kind gift from Dr. Erik Holmgren).
Proper citation: RRID:Addgene_137730 Copy
Species: Homo sapiens
Genetic Insert: CCR5 Promoter
Vector Backbone Description: Backbone Marker:Promega; Vector Backbone:pGL-3 Basic Vector; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:28331180
Comments: pGL-3 basic vector (Promega) can be used as a control to provide baseline luciferase activity
Proper citation: RRID:Addgene_137699 Copy
Species: Salmonella enterica serovar Typhimurium
Genetic Insert: SopF
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:5847; Vector Backbone:pEGFP-C1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_137734 Copy
Species: Mus musculus
Genetic Insert: TKR2/4 microRNA
Vector Backbone Description: Backbone Marker:Åkerblom et al., 2013; Vector Backbone:pLV-PGK-GFP; Vector Types:Mammalian Expression, Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30033154
Comments: the website for the BLOCK-iT RNAi Designer
Proper citation: RRID:Addgene_137733 Copy
Species: Saccharomyces cerevisiae
Genetic Insert: yeTadA1.0-T7 RNAP
Vector Backbone Description: Backbone Size:5600; Vector Backbone:ColE1/AMP; Vector Types:Bacterial Expression, Yeast Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:33707425
Comments: Depositor confirms E366G does not affect plasmid function.
Proper citation: RRID:Addgene_137735 Copy
Species: Saccharomyces cerevisiae
Genetic Insert: RPL25-U1A-PGK1 3' UTR
Vector Backbone Description: Backbone Size:5726; Vector Backbone:pRS426; Vector Types:Yeast Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:11142374
Comments: RPL25 promoter and RPL25 ORF (XhoI/BamHI fragment), U1A hairpins (BamHI/BglII fragment), PGK1 3' UTR (SpeI/NotI fragment), ADH2 terminator (NotI/SacII fragment), 2 micron plasmid.
Proper citation: RRID:Addgene_1375 Copy
Species: Homo sapiens
Genetic Insert: U1A (1-94)
Vector Backbone Description: Backbone Size:4967; Vector Backbone:pRS313; Vector Types:Yeast Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:11142374
Comments: CEN plasmid, GAL1 promoter
Proper citation: RRID:Addgene_1376 Copy
Species: na
Genetic Insert: pGAL-REV-GFP
Vector Backbone Description: Backbone Size:0; Vector Backbone:na; Vector Types:Yeast Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:9636166
Comments: 350 bp SalI-PstI fragment containing Rev from pPS1493 in PstI-XhoI pPS904.
Proper citation: RRID:Addgene_1377 Copy
Species: Clostridioides difficile
Genetic Insert: srtB-linker-hibitopt
Vector Backbone Description: Backbone Marker:Fagan and Fairweather; Backbone Size:7176; Vector Backbone:pRPF185; Vector Types:Bacterial Expression, Luciferase; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:32868401
Proper citation: RRID:Addgene_137753 Copy
Species: Homo sapiens
Genetic Insert: MAP1LC3C
Vector Backbone Description: Vector Backbone:pRK5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31971854
Proper citation: RRID:Addgene_137758 Copy
Species: Homo sapiens
Genetic Insert: MAP1LC3B
Vector Backbone Description: Vector Backbone:pRK5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31971854
Comments: 3' codons have been altered from original reference sequence, while preserving the amino acid sequence.
Proper citation: RRID:Addgene_137757 Copy
Species: Homo sapiens
Genetic Insert: GABARAPL1
Vector Backbone Description: Vector Backbone:pRK5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31971854
Proper citation: RRID:Addgene_137759 Copy
Species: Homo sapiens
Genetic Insert: GABARAP
Vector Backbone Description: Vector Backbone:pRK5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31971854
Proper citation: RRID:Addgene_137760 Copy
Species: Other
Genetic Insert: GFP
Vector Backbone Description: Vector Backbone:pRK5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31971854
Proper citation: RRID:Addgene_137763 Copy
Species: Homo sapiens
Genetic Insert: CALCOCO1
Vector Backbone Description: Vector Backbone:pRK5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31971854
Proper citation: RRID:Addgene_137765 Copy
Species: Homo sapiens
Genetic Insert: CALCOCO1
Vector Backbone Description: Vector Backbone:pRK5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31971854
Proper citation: RRID:Addgene_137766 Copy
Species: Homo sapiens
Genetic Insert: CALCOCO1 W47A
Vector Backbone Description: Vector Backbone:pCMV6-Entry; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:31971854
Proper citation: RRID:Addgene_137769 Copy
Genetic Insert: Rhodopsin promoter-Cre
Vector Backbone Description: Backbone Size:5362; Vector Backbone:pCAGEN?; Vector Types:Mammalian Expression, Cre/Lox; Bacterial Resistance:Ampicillin
Defining Citation: PMID:17209010
Comments: The rhodopsin promoter-Cre is specific for photorecetor cells in the retina.
Cre has a Myc-tag at its C-terminus.
Proper citation: RRID:Addgene_13779 Copy
Species: Bos taurus
Genetic Insert: Bovine GRK2
Vector Backbone Description: Vector Backbone:pEGFP-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:32095688
Proper citation: RRID:Addgene_137778 Copy
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