Basic Vector Information
Lenti-dCas9-KRAB-blast 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-dCas9-KRAB-blast vector Sequence
LOCUS Lenti-dCas9-KRAB 14229 bp DNA circular SYN 09-SEP-2021
DEFINITION Plasmid expression dCas9 protein in fusion with KRAB domain.
ACCESSION .
VERSION .
KEYWORDS Lenti-dCas9-KRAB-blast.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14229)
AUTHORS Xie S, Duan J, Li B, Zhou P, Hon GC
TITLE Multiplexed Engineering and Analysis of Combinatorial Enhancer
Activity in Single Cells.
JOURNAL Mol Cell. 2017 Apr 20;66(2):285-299.e5. doi:
10.1016/j.molcel.2017.03.007. Epub 2017 Apr 13.
PUBMED 28416141
REFERENCE 2 (bases 1 to 14229)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14229)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.molcel.2017.03.007"; journalName: "Mol Cell"; date:
"2017-04-20- 20"; volume: "66"; issue: "2"; pages: "285-299.e5"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14229
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 18..347
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
promoter 395..442
/label=EM7 promoter
/note="synthetic bacterial promoter"
CDS 461..832
/label=BleoR
/note="antibiotic-binding protein"
polyA_signal 965..1098
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1135..1151)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(1159..1175)
/label=lac operator
/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(1183..1213)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(1228..1249)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(1366..1383)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(1537..2125)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2299..3156)
/label=AmpR
/note="beta-lactamase"
promoter complement(3157..3261)
/label=AmpR promoter
primer_bind complement(3336..3355)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 3527..3906
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 3907..4109
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 4124..4304
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 4351..4476
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 4969..5202
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 5387..5431
/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 5580..5621
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
misc_feature 5729..5846
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 5976..7154
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 7170..7217
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 7218..11318
/label=dCas9
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 11373..11393
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 11436..11630
/label=KRAB
/note="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
CDS 11697..11717
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 11733..11786
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 11787..12182
/label=BSD
/note="blasticidin S deaminase"
misc_feature 12210..12798
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(12801..12817)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12802..12818)
/label=pBluescriptKS
/note="For pBluescript vector"
LTR 13323..13503
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 13535..13759
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 13805..14229
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
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