Basic Vector Information
- Vector Name:
- pLV3-U6-MCS2-sgRNA-Cas9-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 12194 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-MCS2-sgRNA-Cas9-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-MCS2-sgRNA-Cas9-Puro vector Sequence
LOCUS V015052 12194 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V015052
VERSION V015052
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 12194)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..12194
/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 4821..4874
/label="T2A"
/note="2A peptide from Thosea asigna virus capsid protein"
CDS 4875..5471
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 5497..6085
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 6157..6390
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 6422..6646
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 6692..7120
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
polyA_signal 7263..7396
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
promoter complement(7481..7511)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(7526..7547)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(7835..8423)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8597..9454)
/label="AmpR"
/note="beta-lactamase"
promoter complement(9455..9559)
/label="AmpR promoter"
promoter 10205..10407
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 10422..10602
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 10649..10774
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 11267..11500
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 11685..11729
/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 11878..11919
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 12027..12144
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
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