Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V011557 | pCMV-Cypridina Luc | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
This control vector enables constitutive, high-level expression of secreted Cypridina luciferase in mammalian cells, driven by the strong CMV promoter for robust reporter activity
- Vector Name:
- pCMV-Cypridina Luc
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6586 bp
- Type:
- Luciferase Vectors
- Replication origin:
- ori
- Source/Author:
- Thermo Scientific (Pierce)
- Copy Number:
- High copy number
- Promoter:
- SV40
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pCMV-Cypridina Luc vector Map
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
- Lee MC, Huang HJ, Chang TH, Huang HC, Hsieh SY, Chen YS, Chou WY, Chiang CH, Lai CH, Shiau CY. Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions. Sci Rep. 2016 Jul 4;6:29311. doi: 10.1038/srep29311. PMID: 27373565; PMCID: PMC4931692.
pCMV-Cypridina Luc vector Sequence
LOCUS 40924_11621 6586 bp DNA circular SYN 01-JAN-1980
DEFINITION Control vector for constitutive high-level expression of secreted
Cypridina luciferase under control of the CMV promoter.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6586)
AUTHORS Thermo Scientific (Pierce)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 6586)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6586
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 63..366
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 367..570
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 615..633
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 646..2304
/codon_start=1
/label=CLuc
/note="secreted Cypridina luciferase"
/translation="MKTLILAVALVYCATVHCQDCPYEPDPPNTVPTSCEAKEGECIDS
SCGTCTRDILSDGLCENKPGKTCCRMCQYVIECRVEAAGWFRTFYGKRFQFQEPGTYVL
GQGTKGGDWKVSITLENLDGTKGAVLTKTRLEVAGDIIDIAQATENPITVNGGADPIIA
NPYTIGEVTIAVVEMPGFNITVIEFFKLIVIDILGGRSVRIAPDTANKGMISGLCGDLK
MMEDTDFTSDPEQLAIQPKINQEFDGCPLYGNPDDVAYCKGLLEPYKDSCRNPINFYYY
TISCAFARCMGGDERASHVLLDYRETCAAPETRGTCVLSGHTFYDTFDKARYQFQGPCK
EILMAADCFWNTWDVKVSHRNVDSYTEVEKVRIRKQSTVVELIVDGKQILVGGEAVSVP
YSSQNTSIYWQDGDILTTAILPEALVVKFNFKQLLVVHIRDPFDGKTCGICGNYNQDFS
DDSFDAEGACDLTPNPPGCTEEQKPEAERLCNSLFAGQSDLDQKCNVCHKPDRVERCMY
EYCLRGQQGFCDHAWEFKKECYIKHGDTLEVPDECK"
polyA_signal 2330..2441
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 2564..2893
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
promoter 2941..2988
/label=EM7 promoter
/note="synthetic bacterial promoter"
CDS 3007..3603
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
polyA_signal 3736..3869
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
CDS complement(3913..4770)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5034..5622
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5892..5908
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 6364..6380
/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)."
misc_feature 6495..6586
/label=pause site
/note="RNA polymerase II transcriptional pause signal from
the human alpha-2 globin gene"