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pCS2+HyPer7 vector (V001192)

Price Information

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V001192 pCS2+HyPer7 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

Mammalian expression of non-targeted ultrasensitive hydrogen peroxide indicator HyPer7 for optical imaging. HyPer7 is a hydrogen peroxide probe based on cpGFP integrated into the OxyR-RD domain from Neisseria meningitidis.

Vector Name:
pCS2+HyPer7
Antibiotic Resistance:
Ampicillin
Length:
5531 bp
Type:
Mammalian Expression
Replication origin:
ori
Source/Author:
Vsevolod Belousov
Copy Number:
High Copy
Promoter:
sCMV
Cloning Method:
Restriction Enzyme
5' Primer:
ATTTAGGTGACACTATAGA
3' Primer:
GTCATAGCTGTTTCCTG
Growth Strain(s):
DH5alpha
Growth Temperature:
37℃

pCS2+HyPer7 vector Map

Copy Sequence View Map
pCS2+HyPer75531 bp60012001800240030003600420048005400SP6 promoteroxyRcpGFPoxyRT7 promoterSV40 poly(A) signalT3 promoterM13 revlac operatorlac promoterCAP binding siteL4440oriAmpRAmpR promoterf1 oriCMV IE94 promoter

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.

References

  • Pak VV, Ezeriņa D, Lyublinskaya OG, Pedre B, Tyurin-Kuzmin PA, Mishina NM, Thauvin M, Young D, Wahni K, Martínez Gache SA, Demidovich AD, Ermakova YG, Maslova YD, Shokhina AG, Eroglu E, Bilan DS, Bogeski I, Michel T, Vriz S, Messens J, Belousov VV. Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function. Cell Metab. 2020 Mar 3;31(3):642-653.e6. doi: 10.1016/j.cmet.2020.02.003. PMID: 32130885; PMCID: PMC7088435.

pCS2+HyPer7 vector Sequence

LOCUS       Exported                5531 bp DNA     circular SYN 31-JUL-2024
DEFINITION  Mammalian expression of non-targeted ultrasensitive hydrogen 
            peroxide indicator HyPer7 for optical imaging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5531)
  AUTHORS   Pak VV, Ezerina D, Lyublinskaya OG, Pedre B, Tyurin-Kuzmin PA, 
            Mishina NM, Thauvin M, Young D, Wahni K, Martinez Gache SA, 
            Demidovich AD, Ermakova YG, Maslova YD, Shokhina AG, Eroglu E, Bilan
            DS, Bogeski I, Michel T, Vriz S, Messens J, Belousov VV
  TITLE     Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide 
            Identifies Roles for the Oxidant in Cell Migration and Mitochondrial
            Function.
  JOURNAL   Cell Metab. 2020 Mar 3;31(3):642-653.e6. doi: 
            10.1016/j.cmet.2020.02.003.
  PUBMED    32130885
REFERENCE   2  (bases 1 to 5531)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5531)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5531)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cmet.2020.02"; journalName: "Cell Metab"; date: 
            "2020-03-3- 3"; volume: "31"; issue: "3"; pages: "642-653.e6"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5531
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     CDS             106..489
                     /codon_start=1
                     /product="OxyR-RD domain from Neisseria meningitidis"
                     /label=oxyR
                     /translation="MHLANEEQNELEGAFKLGLIFTVAPYLLPKLIVSLRRTAPKMPLM
                     LEENYTHTLTESLKRGDVDAIIVAEPFQEPGIVTEPLYDEPFFVIVPKGHSFEELDAVS
                     PRMLGEEQVLLLTEGNCMRDQVLS"
     CDS             511..1236
                     /codon_start=1
                     /label=cpGFP
                     /note="circularly permuted GFP"
                     /translation="NVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPV
                     LLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLS
                     YGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIGFKEDGNILGHKLEYN"
     primer_bind     717..738
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(847..868)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1108..1127)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1243..1542
                     /codon_start=1
                     /product="OxyR-RD domain from Neisseria meningitidis"
                     /label=oxyR
                     /translation="SCSELAAKQRIQGLTNTLQGSSINTIRHMVASGLAISVLPATALT
                     ENDHMLFSIIPFEGTPPSRRVVLAYRRNFVRPKALSAMKAAIMQSQLHGVSFICD"
     promoter        complement(1561..1578)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    complement(1583..1717)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(1830..1848)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1869..1885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(1893..1909)
                     /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(1917..1947)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(1962..1983)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(2100..2117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2271..2859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3033..3890)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(3891..3995)
                     /label=AmpR promoter
     rep_origin      complement(4021..4476)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4553..5531
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus
                     major immediate early transcription unit IE94"