Basic Vector Information
lentiCas9-EGFP 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 ℃.
lentiCas9-EGFP vector Sequence
LOCUS lentiCas9-EGFP. 13177 bp DNA circular SYN 01-JAN-1980 DEFINITION Zhang lab lentiviral vector for expressing human codon-optimized S. pyogenes Cas9 plus enhanced GFP (EGFP). ACCESSION . VERSION . KEYWORDS lentiCas9-EGFP. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13177) AUTHORS Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, Scott DA, Song J, Pan JQ, Weissleder R, Lee H, Zhang F, Sharp PA. TITLE Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. JOURNAL Cell 2015;160:1246-60. PUBMED 25748654 REFERENCE 2 (bases 1 to 13177) AUTHORS TITLE Direct Submission REFERENCE 3 (bases 1 to 13177) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell"; date: "2015"; volume: "160"; pages: "1246-60" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..13177 /mol_type="other DNA" /organism="synthetic DNA construct" enhancer 238..617 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 618..820 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" LTR 835..1015 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1680..1913 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2098..2142 /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 2291..2332 /note="Protein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509" misc_feature 2440..2557 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2650..2861 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 2886..6989 /label=Cas9 /note="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" CDS 6990..7037 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=bipartite nuclear localization signal from nucl /note="nucleoplasmin NLS" /translation="KRPAATKKAGQAKKKK" CDS 7038..7061 /label=FLAG /note="FLAG(R) epitope tag, followed by an enterokinase cleavage site" CDS 7071..7127 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=2A peptide from porcine teschovirus-1 polyprotein /note="P2A" /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 7128..7841 /label=EGFP /note="enhanced GFP" misc_feature 7869..8457 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" LTR 8982..9162 /label=5' LTR (truncated) /note="5' LTR (truncated)" /note="truncated 5' long terminal repeat (LTR) from HIV-1" polyA_signal 9194..9418 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 9464..9892 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 10017..10227 /label=SV40 promoter /note="SV40 early promoter" promoter 10283..10330 /label=EM7 promoter /note="synthetic bacterial promoter" CDS 10349..10720 /label=BleoR /note="antibiotic-binding protein" polyA_signal 10853..10986 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" promoter complement(11071..11101) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(11116..11137) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(11425..12013) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(12187..13044) /label=AmpR /note="beta-lactamase" promoter complement(13045..13149) /label=AmpR promoter
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