Basic Vector Information
- Vector Name:
- Fuw-dCas9-Dnmt3a_IM
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14241 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Selection Marker:
- Zeocin ; Resistance marker is outside the LTRs and will not be packaged into virus
- Copy Number:
- High Copy
- Promoter:
- UbC
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- aactatgcgctcggg
- 3' Primer:
- taaagcagcgtatcc
Fuw-dCas9-Dnmt3a_IM 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 ℃.
Fuw-dCas9-Dnmt3a_IM vector Sequence
LOCUS Fuw-dCas9-Dnmt3a 14241 bp DNA circular SYN 13-MAY-2021
DEFINITION Lentiviral construct to express dCas9 fused with an inactive Dnmt3a
catalytic domain (E664A, E756A).
ACCESSION .
VERSION .
KEYWORDS Fuw-dCas9-Dnmt3a_IM.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14241)
AUTHORS Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S, Shu J, Dadon D,
Young RA, Jaenisch R
TITLE Editing DNA Methylation in the Mammalian Genome.
JOURNAL Cell. 2016 Sep 22;167(1):233-247.e17. doi:
10.1016/j.cell.2016.08.056.
PUBMED 27662091
REFERENCE 2 (bases 1 to 14241)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14241)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.cell.2016.08"; journalName: "Cell"; date: "2016-09-22-
22"; volume: "167"; issue: "1"; pages: "233-247.e17"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14241
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 91..470
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 471..673
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 688..868
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 915..1040
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1533..1766
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1951..1995
/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 2144..2185
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
misc_feature 2293..2410
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2475..3686
/label=UbC promoter
/note="human ubiquitin C promoter"
CDS 3737..7840
/label=dCas9
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 7853..7873
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7877..7897
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 7901..7921
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 7925..8830
/label=DNMT3A catalytic domain
/note="catalytic domain of DNA methyltransferase 3A"
misc_feature 8858..9446
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(9449..9465)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9450..9466)
/label=pBluescriptKS
/note="For pBluescript vector"
LTR 9971..10151
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 10183..10407
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 10453..10881
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(10890..10910)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 10934..11144
/label=SV40 promoter
/note="SV40 early promoter"
promoter 11200..11247
/label=EM7 promoter
/note="synthetic bacterial promoter"
CDS 11266..11637
/label=BleoR
/note="antibiotic-binding protein"
polyA_signal 11770..11903
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(11940..11956)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11940..11956)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11953..11975)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 11964..11980
/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(11988..12018)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(12033..12054)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12171..12188)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(12342..12930)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13104..13961)
/label=AmpR
/note="beta-lactamase"
promoter complement(13962..14066)
/label=AmpR promoter
primer_bind complement(14141..14160)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
This page is informational only.