Fuw-dCas9-Dnmt3a_IM vector (V003719)

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

Fuw-dCas9-Dnmt3a_IM14241 bp7001400210028003500420049005600630070007700840091009800105001120011900126001330014000CMV enhancerCMV promoter5' LTR (truncated)HIV-1 PsiRREgp41 peptideProtein TatcPPT/CTSUbC promoterdCas9SV40 NLSSV40 NLSSV40 NLSDNMT3A catalytic domainWPREKS primer5' LTR (truncated)bGH poly(A) signalf1 oripBABE 3'SV40 promoterEM7 promoterBleoRSV40 poly(A) signalIn lacZ genelac promoterCAP binding siteL4440oriAmpRAmpR promoterpRS-marker

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

Copy Sequence

Download GenBank File(.gb)

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"