Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V014948 | pDSK-GFPuv-Cam | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The plasmid could be transferred into various bacteria by electroporation using standard protocols or triparental mating using pRK2013 as a helper plasmid. GFPuv expression is driven by a constitutive chloroplast promoter psbA at high levels.
- Vector Name:
- pDSK-GFPuv-Cam
- Antibiotic Resistance:
- Kanamycin
- Length:
- 10400 bp
- Type:
- Protein expression
- Replication origin:
- RSF1010 oriV
- Host:
- Broad host
- Selection Marker:
- Chl
- Growth Strain(s):
- stbl3
- Growth Temperature:
- 37℃
pDSK-GFPuv-Cam 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
- Wang Z, Liu Y, Li L, Li D, Zhang Q, Guo Y, Wang S, Zhong C, Huang H. Whole transcriptome sequencing of Pseudomonas syringae pv. actinidiae-infected kiwifruit plants reveals species-specific interaction between long non-coding RNA and coding genes. Sci Rep. 2017 Jul 7;7(1):4910. doi: 10.1038/s41598-017-05377-y. PMID: 28687784; PMCID: PMC5501815.
pDSK-GFPuv-Cam vector Sequence
LOCUS pDSK-GFPuv-Cam 10400 bp DNA circular SYN 22-SEP-2024
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10400)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..10400
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 1079..1095
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
CDS complement(1102..1761)
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQSGA"
promoter complement(1762..1864)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
RBS 2071..2093
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 2101..2817
/codon_start=1
/product="GFP variant optimized for excitation by UV light"
/label=GFPuv
/translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITHGMDELYK"
primer_bind complement(2866..2882)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2890..2906
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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(2914..2944)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2959..2980
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(3644..4495)
/codon_start=1
/gene="repC"
/product="replication protein C of the broad-host-range
plasmid RSF1010 (Scholz et al., 1989)"
/label=RSF1010 RepC
/translation="MVKPKNKHSLSHVRHDPAHCLAPGLFRALKRGERKRSKLDVTYDY
GDGKRIEFSGPEPLGADDLRILQGLVAMAGPNGLVLGPEPKTEGGRQLRLFLEPKWEAV
TADAMVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
TGKASIDTLCGYVWPSEASGSTMRKRRQRVREALPELVALGWTVTEFAAGKYDITRPKA
AG"
CDS complement(4482..5321)
/codon_start=1
/gene="repA"
/product="replication protein A of the broad-host-range
plasmid RSF1010 (Scholz et al., 1989)"
/label=RSF1010 RepA
/translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
CDS complement(5832..6803)
/codon_start=1
/gene="repB"
/product="replication protein B of the broad-host-range
plasmid RSF1010 (Scholz et al., 1989)"
/label=RSF1010 RepB
/translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
oriT 8042..8129
/label=RSF1010 oriT
/note="origin of transfer of the broad-host-range plasmid
RSF1010 (Scholz et al., 1989)"
rep_origin 8471..8865
/label=RSF1010 oriV
/note="replication origin of the broad-host-range plasmid
RSF1010; requires the RSF1010 RepA/B/C proteins for
replication (Scholz et al., 1989)"
CDS complement(join(9679..10400,1..94))
/codon_start=1
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"