Basic Vector Information
- Vector Name:
- pLV3-U6-MCS-Stuffer-sgRNA-Cas9-EGFP-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 13194 bp
- Type:
- Genome editing
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro
- Promoter:
- U6
- 5' Primer:
- U6-F
- Growth Temperature:
- 37℃
pLV3-U6-MCS-Stuffer-sgRNA-Cas9-EGFP-Puro 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 ℃.
pLV3-U6-MCS-Stuffer-sgRNA-Cas9-EGFP-Puro vector Sequence
LOCUS 62056_15735 13194 bp DNA circular SYN 01-JAN-1980 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13194) AUTHORS . TITLE Direct Submission FEATURES Location/Qualifiers source 1..13194 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 489..564 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 626..837 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 862..4965 /label=Cas9 /note="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" CDS 5014..5037 /label=FLAG /note="FLAG(R) epitope tag, followed by an enterokinase cleavage site" CDS 5104..5820 /label=EGFP /note="enhanced GFP" misc_feature 6497..7085 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" LTR 7157..7390 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 7422..7646 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 7692..8120 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" polyA_signal 8263..8396 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" promoter complement(8481..8511) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(8526..8547) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(8835..9423) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(9597..10454) /label=AmpR /note="beta-lactamase" promoter complement(10455..10559) /label=AmpR promoter enhancer 10825..11204 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 11205..11407 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" LTR 11422..11602 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 11649..11774 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 12267..12500 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 12685..12729 /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 12878..12919 /note="Protein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509" misc_feature 13027..13144 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1"
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