Basic Vector Information
Lenti-(BB)-EF1a-KRAB-dCas9-P2A-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 ℃.
Lenti-(BB)-EF1a-KRAB-dCas9-P2A-EGFP vector Sequence
LOCUS Lenti-(BB)-EF1a- 15284 bp DNA circular SYN 13-MAY-2021
DEFINITION Catalytically inactive Cas9 from S. pyogenes with P2A-EGFP under the
EF1a core promoter, and cloning backbone for sgRNA. Contains BsmBI
sites for insertion of spacer sequences..
ACCESSION .
VERSION .
KEYWORDS Lenti-(BB)-EF1a-KRAB-dCas9-P2A-EGFP.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15284)
TITLE Ferrer Lab Plasmids
REFERENCE 2 (bases 1 to 15284)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 15284)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..15284
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter complement(176..280)
/label=AmpR promoter
enhancer 546..925
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 926..1128
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 1143..1323
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1370..1495
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1988..2221
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2406..2450
/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 2599..2640
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
misc_feature 2748..2865
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2916..3156
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 5046..5121
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 5183..5394
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 5650..9753
/label=dCas9
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 9946..10659
/label=EGFP
/note="enhanced GFP"
misc_feature 10684..11272
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 11344..11577
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 11609..11833
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 11879..12307
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 12321..12650
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
promoter 12698..12745
/label=EM7 promoter
/note="synthetic bacterial promoter"
CDS 12764..13135
/label=BleoR
/note="antibiotic-binding protein"
polyA_signal 13268..13401
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
promoter complement(13486..13516)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(13531..13552)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(13840..14428)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(join(14602..15284,1..175))
/label=AmpR
/note="beta-lactamase"
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