Basic Vector Information
- Vector Name:
- CRISPRi library backbone
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8888 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead
- Copy Number:
- High copy number
- Promoter:
- EF-1α
CRISPRi library backbone vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
CRISPRi library backbone vector Sequence
LOCUS CRISPRi_library 8888 bp DNA circular SYN 01-JAN-1980 DEFINITION Backbone vector for generating a CRISPR-mediated transcriptional repression (CRISPRi) library. ACCESSION . VERSION . KEYWORDS CRISPRi library backbone. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8888) AUTHORS Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M, Weissman JS. TITLE Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. JOURNAL Cell 2014;159:647-61. PUBMED 25307932 REFERENCE 2 (bases 1 to 8888) AUTHORS Weissman Lab / Addgene #62217 TITLE Direct Submission REFERENCE 3 (bases 1 to 8888) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell"; date: "2014"; volume: "159"; pages: "647-61" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..8888 /mol_type="other DNA" /organism="synthetic DNA construct" enhancer 247..626 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 627..829 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" LTR 844..1024 /label=LTR (truncated) /note="LTR (truncated)" /note="truncated long terminal repeat (LTR) from HIV-1" misc_feature 1071..1196 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1695..1928 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2113..2157 /label=gp41 peptide /note="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" CDS 2306..2347 /note="Protein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509" misc_feature 2455..2572 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2631..2943 /note="U6 promoter" /note="modified RNA polymerase III promoter for mouse U6 snRNA" protein_bind 2825..2858 /label=Cre recombinase binding site /bound_moiety="Cre recombinase" /note="loxP" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 2965..3050 /label=gRNA scaffold /note="gRNA scaffold" /note="modified guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 3051..3056 /note="polIII terminator" /note="RNA polymerase III transcription terminator" promoter 3114..4286 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" CDS 4298..4894 /label=PuroR /note="puromycin N-acetyltransferase" CDS 4904..4957 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=2A peptide from Thosea asigna virus capsid protein /note="T2A" /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 4970..5665 /label=TagBFP /note="monomeric blue fluorescent protein" protein_bind complement(5688..5721) /label=loxP /note="Cre-mediated recombination occurs in the 8-bp core sequence (ATGTATGC) (Shaw et al., 2021)." misc_feature 5777..6365 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(6368..6384) /label=KS primer /note="common sequencing primer, one of multiple similar variants" LTR 6894..7074 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(7136..7724) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(7898..8755) /label=AmpR /note="beta-lactamase" promoter complement(8756..8860) /label=AmpR promoter
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