Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V003719 | Fuw-dCas9-Dnmt3a_IM | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
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 Map
Plasmid 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 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
Fuw-dCas9-Dnmt3a_IM vector Sequence
LOCUS V003719 14241 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V003719
VERSION V003719
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"
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"
/label="Protein Tat"
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"