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Flag-HA-USP36 vector (V017267)

Price Information

Cat No. Plasmid Name Availability Add to cart
V017267 Flag-HA-USP36 In stock, 1 week for quality controls

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Vector Name:
Flag-HA-USP36
Antibiotic Resistance:
Ampicillin
Length:
9812 bp
Type:
Gene expression
Replication origin:
ori
Host:
Mammalian cells, Retrovirus
Selection Marker:
Puro
Promoter:
MSCV
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

Flag-HA-USP36 vector Map

Copy Sequence View Map
Flag-HA-USP369812 bp4008001200160020002400280032003600400044004800520056006000640068007200760080008400880092009600oriAmpRAmpR promoter5' LTRMESV Psigag (truncated)FLAGHAUbiquitin carboxyl-terminal hydrolase 36attB2IRES2PuroR3' LTRlac operatorlac promoterCAP binding site

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.

Flag-HA-USP36 vector Sequence

LOCUS       V017267                 9812 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V017267
VERSION     V017267
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 9812)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(234..822)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(996..1853)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(1854..1958)
                     /label="AmpR promoter"
     LTR             2786..3302
                     /label="5' LTR"
                     /note="5' long terminal repeat from murine embryonic stem
                     cell virus"
     misc_feature    3366..3707
                     /label="MESV Psi"
                     /note="packaging signal of murine embryonic stem cell
                     virus"
     CDS             3774..4190
                     /label="gag (truncated)"
                     /note="truncated Moloney murine leukemia virus (MMLV) gag
                     gene lacking the start codon"
     CDS             4212..4235
                     /label="FLAG"
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             4248..4274
                     /label="HA"
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             4323..7691
                     /gene="USP36"
                     /label="Ubiquitin carboxyl-terminal hydrolase 36"
                     /note="Ubiquitin carboxyl-terminal hydrolase 36 from Homo
                     sapiens. Accession#: Q9P275"
     protein_bind    complement(7702..7722)
                     /label="attB2"
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     misc_feature    7740..8316
                     /label="IRES2"
                     /note="internal ribosome entry site (IRES) of the
                     encephalomyocarditis virus (EMCV)"
     CDS             8317..8913
                     /label="PuroR"
                     /note="puromycin N-acetyltransferase"
     LTR             8992..9506
                     /label="3' LTR"
                     /note="3' long terminal repeat from murine embryonic stem
                     cell virus"
     protein_bind    complement(9668..9684)
                     /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(9692..9722)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(9737..9758)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."