Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V010264 | pGBKT7 | In stock, 1 week for quality controls |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pGBKT7
- Antibiotic Resistance:
- Kanamycin
- Length:
- 7303 bp
- Type:
- Yeast Plasmids
- Replication origin:
- ori
- Source/Author:
- Clontech
- Selection Marker:
- Neomycin/G418(Geneticin)
- Copy Number:
- High copy number
- Promoter:
- ADH1
- 3' Primer:
- M13 rev
pGBKT7 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.
pGBKT7 vector Sequence
LOCUS Exported 7303 bp DNA circular SYN 09-SEP-2012
DEFINITION Yeast two-hybrid ""bait"" vector for expressing proteins fused to
the GAL4 DNA-binding domain.
ACCESSION .
VERSION .
KEYWORDS pGBKT7
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7303)
AUTHORS Clontech
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7303
/lab_host="Saccharomyces cerevisiae"
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 30..734
/gene="S. cerevisiae ADH1"
/label=ADH1 promoter
/note="promoter for alcohol dehydrogenase 1"
CDS 762..1202
/codon_start=1
/gene="Saccharomyces cerevisiae GAL4 (truncated)"
/product="DNA binding domain of the GAL4 transcriptional
activator"
/label=GAL4 DNA binding domain
/translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
promoter 1213..1231
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1245..1247
/codon_start=1
/product="start codon for in vitro
transcription/translation"
/label=ATG
/translation="M"
CDS 1251..1280
/codon_start=1
/product="Myc (human c-Myc oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
misc_feature 1281..1333
/label=MCS
/note="multiple cloning site"
terminator 1338..1385
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
terminator 1412..1599
/gene="S. cerevisiae ADH1"
/label=ADH1 terminator
/note="transcription terminator for alcohol dehydrogenase
1"
primer_bind complement(1623..1639)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 1647..1663
/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(1671..1701)
/label=lac promoter
/note="promoter for the E. coli lac operon"
rep_origin complement(2025..2613)
/direction=LEFT
/label=ori
/note="high-copy-number colE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 2942..2989
/label=HSV TK poly(A) signal
/note="herpesvirus thymidine kinase polyadenylation signal"
CDS complement(3221..4015)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin)"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(4016..4120)
/gene="bla"
/label=AmpR promoter
rep_origin 4147..5489
/label=2u ori
/note="yeast 2u plasmid origin of replication"
promoter 5748..6029
/gene="S. cerevisiae TRP1"
/label=TRP1 promoter
CDS 6030..6704
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/label=TRP1
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
rep_origin complement(6807..7262)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"