Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V014565 | pDHtsk-GFP-G418 | 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:
- pDHtsk-GFP-G418
- Antibiotic Resistance:
- Kanamycin
- Length:
- 11518 bp
- Type:
- Gene expression
- Replication origin:
- ori
- Host:
- Hyphal fungi
- Selection Marker:
- Neo/G418
- Promoter:
- NOS
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
pDHtsk-GFP-G418 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.
pDHtsk-GFP-G418 vector Sequence
LOCUS 62056_8445 11518 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11518)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..11518
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 191..209
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 294..312
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 654..837
/label=NOS promoter
/note="nopaline synthase promoter"
CDS 861..1649
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase from Tn5"
/translation="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
LAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
terminator 2042..2294
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(2913..3626)
/codon_start=1
/label=yeGFP
/note="yeast-enhanced green fluorescent protein"
/translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITHGMDELYK"
promoter complement(4434..4452)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter complement(4521..4552)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(4567..4588)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
misc_feature 4956..4980
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 6280..6906
/codon_start=1
/label=pVS1 StaA
/note="stability protein from the Pseudomonas plasmid pVS1
(Heeb et al., 2000)"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 7343..8407
/codon_start=1
/label=pVS1 RepA
/note="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
MRYRNLIEGEASAGS"
rep_origin 8476..8670
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 9014..9154
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(9340..9928)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(10018..10809)
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 11234..11258
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal complement(11336..11510)
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"