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HLTV-hTRIM21 vector (V000027)

Price Information

Cat No. Plasmid Name Availability Add to cart
V000027 HLTV-hTRIM21 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:
HLTV-hTRIM21
Antibiotic Resistance:
Ampicillin
Length:
4456 bp
Type:
Bacterial Expression
Replication origin:
ori
Promoter:
T7
Cloning Method:
Restriction Enzyme
5' Primer:
TAA TAC GAC TCA CTA TAG GG

HLTV-hTRIM21 vector Map

Copy Sequence View Map
HLTV-hTRIM214456 bp600120018002400300036004200pBR322ori-FL4440T7 promoterRBS6xHisTEV siteE3 ubiquitin-protein ligase TRIM21T7 terminatorf1 oriAmpR promoterAmpRori

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.

HLTV-hTRIM21 vector Sequence

LOCUS       V000027                 4456 bp    DNA     circular SYN 09-SEP-2021
DEFINITION  Exported.
ACCESSION   V000027
VERSION     V000027
KEYWORDS    HLTV-hTRIM21
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 4456)
  AUTHORS   Clift D, McEwan WA, Labzin LI, Konieczny V, Mogessie B, James LC,
            Schuh M
  TITLE     A Method for the Acute and Rapid Degradation of Endogenous Proteins.
  JOURNAL   Cell. 2017 Nov 13. pii: S0092-8674(17)31255-2. doi:
            10.1016/j.cell.2017.10.033.
   PUBMED   29153837
REFERENCE   2  (bases 1 to 4456)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4456)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2017
            Nov 13. pii: S0092-8674(17)31255-2. doi:
            10.1016/j.cell.2017.10.033."
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4456
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     37..56
                     /label="pBR322ori-F"
                     /note="pBR322 origin, forward primer"
     primer_bind     290..307
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     promoter        392..410
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             442..464
                     /label="RBS"
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             475..492
                     /label="6xHis"
                     /note="6xHis affinity tag"
     CDS             769..789
                     /label="TEV site"
                     /note="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
     CDS             796..2220
                     /gene="TRIM21"
                     /label="E3 ubiquitin-protein ligase TRIM21"
                     /note="E3 ubiquitin-protein ligase TRIM21 from Homo
                     sapiens. Accession#: P19474"
     terminator      2243..2290
                     /label="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     rep_origin      2387..2842
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2868..2972
                     /label="AmpR promoter"
     CDS             2973..3830
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      4004..4456
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"