Basic Vector Information
- Vector Name:
- pLV3-U6-MCS-sgRNA-Cas9-Fluc-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 13901 bp
- Type:
- Genome editing
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro
- Promoter:
- U6
- 5' Primer:
- U6
- Growth Temperature:
- 37℃
pLV3-U6-MCS-sgRNA-Cas9-Fluc-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-sgRNA-Cas9-Fluc-Puro vector Sequence
LOCUS 62056_15655 13901 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 13901)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..13901
/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 266..341
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 403..614
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 639..4742
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 4791..4814
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 4824..4880
/label=P2A
/note="2A peptide from porcine teschovirus-1 polyprotein"
CDS 4881..6527
/label=luciferase
/note="firefly luciferase"
CDS 6528..6581
/label=T2A
/note="2A peptide from Thosea asigna virus capsid protein"
CDS 6582..7178
/label=PuroR
/note="puromycin N-acetyltransferase"
misc_feature 7204..7792
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 7864..8097
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 8129..8353
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 8399..8827
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
polyA_signal 8970..9103
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
promoter complement(9188..9218)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(9233..9254)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(9542..10130)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(10304..11161)
/label=AmpR
/note="beta-lactamase"
promoter complement(11162..11266)
/label=AmpR promoter
enhancer 11532..11911
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 11912..12114
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 12129..12309
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 12356..12481
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 12974..13207
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 13392..13436
/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 13585..13626
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
misc_feature 13734..13851
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
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