Basic Vector Information
- Vector Name:
- LentiRCas9-Lambda2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 13752 bp
- Type:
- Lentiviral, CRISPR
- Replication origin:
- ori
- Promoter:
- U6
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- LKO1.5
- 3' Primer:
- T3
LentiRCas9-Lambda2 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 ℃.
LentiRCas9-Lambda2 vector Sequence
LOCUS LentiRCas9-Lambd 13752 bp DNA circular SYN 13-MAY-2021
DEFINITION Lentiviral transfer vector that carries U6-driven non-targeting
sgRNA using a modified scaffold (Chen et al. Cell 2013) and
CMV-driven PIN-dCas9. Derived from LentiCRISPR v2 (Zhang lab).
ACCESSION .
VERSION .
KEYWORDS LentiRCas9-Lambda2.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13752)
AUTHORS Batra R, Nelles DA, Pirie E, Blue SM, Marina RJ, Wang H, Chaim IA,
Thomas JD, Zhang N, Nguyen V, Aigner S, Markmiller S, Xia G, Corbett
KD, Swanson MS, Yeo GW
TITLE Elimination of Toxic Microsatellite Repeat Expansion RNA by
RNA-Targeting Cas9.
JOURNAL Cell. 2017 Aug 24;170(5):899-912.e10. doi:
10.1016/j.cell.2017.07.010. Epub 2017 Aug 10.
PUBMED 28803727
REFERENCE 2 (bases 1 to 13752)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 13752)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.cell.2017.07.010"; journalName: "Cell"; date:
"2017-08-24- 24"; volume: "170"; issue: "5"; pages: "899-912.e10"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..13752
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 381..583
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 598..778
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 825..950
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1443..1676
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1861..1905
/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 2054..2095
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
misc_feature 2203..2320
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2371..2611
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(2748..2764)
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(2801..2819)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 2881..3092
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 3720..7820
/label=dCas9
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 7824..7850
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 7869..7889
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7896..7916
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7938..7994
/label=P2A
/note="2A peptide from porcine teschovirus-1 polyprotein"
CDS 7995..8588
/label=PuroR
/note="puromycin N-acetyltransferase"
misc_feature 8607..9195
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 9267..9500
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 9532..9756
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 9802..10230
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(9889..9908)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 10099..10120
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 10244..10573
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
promoter 10621..10668
/label=EM7 promoter
/note="synthetic bacterial promoter"
CDS 10687..11058
/label=BleoR
/note="antibiotic-binding protein"
polyA_signal 11191..11324
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(11361..11377)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11361..11377)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11374..11396)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 11385..11401
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(11409..11439)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(11454..11475)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11592..11609)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(11763..12351)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12525..13382)
/label=AmpR
/note="beta-lactamase"
promoter complement(13383..13487)
/label=AmpR promoter
primer_bind complement(13562..13581)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
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