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sgRNA(MS2) cloning backbone vector (V000235)

Price Information

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V000235 sgRNA(MS2) cloning backbone In stock, instant shipping

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

Basic Vector Information

Vector Name:
sgRNA(MS2) cloning backbone
Antibiotic Resistance:
Ampicillin
Length:
3008 bp
Type:
Mammalian Expression, CRISPR
Replication origin:
ori
Copy Number:
High Copy
Promoter:
U6
Cloning Method:
Restriction Enzyme
5' Primer:
CACCTCTGACTTGAGCGTCGATTTTTGTG
3' Primer:
CCCGCCGCGCTTAATGC
Growth Strain(s):
DH10B
Growth Temperature:
37℃

sgRNA(MS2) cloning backbone vector Map

Copy Sequence View Map
sgRNA(MS2) cloning backbone3008 bp6001200180024003000U6 promoterMS2 stem loopMS2 stem loopf1 oripRS-markerpGEX 3'pBRforEcoAmpR 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.

sgRNA(MS2) cloning backbone vector Sequence

LOCUS       40924_48788        3008 bp DNA     circular SYN 13-MAY-2021
DEFINITION  sgRNA cloning backbone with MS2 loops at tetraloop and stemloop 2. 
            Contains BbsI sites for insertion of spacer sequences..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3008)
  AUTHORS   Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena 
            C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F
  TITLE     Genome-scale transcriptional activation by an engineered CRISPR-Cas9
            complex.
  JOURNAL   Nature. 2014 Dec 10. doi: 10.1038/nature14136.
  PUBMED    25494202
REFERENCE   2  (bases 1 to 3008)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3008)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2014 Dec 10. doi: 10.1038/nature14136."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..247
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        291..309
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat
                     protein"
     misc_RNA        361..379
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat
                     protein"
     rep_origin      491..946
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(963..982)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     1082..1104
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1142..1160)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        1228..1332
                     /label=AmpR promoter
     CDS             1333..2190
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2364..2952
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"