Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V005705 | pGWB655 | 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
- Vector Name:
- pGWB655
- Antibiotic Resistance:
- Streptomycin
- Length:
- 12218 bp
- Type:
- Gateway binary vector
- Replication origin:
- ori
- Host:
- Plants
- Source/Author:
- Nakamura S, Mano S, Tanaka Y, Ohnishi M, Nakamori C, Araki M, Niwa T, Nishimura M, Kaminaka H, Nakagawa T, Sato Y, Ishiguro S.
- Promoter:
- CaMV35S(long)
pGWB655 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.
pGWB655 vector Sequence
LOCUS 40924_23594 12218 bp DNA circular SYN 18-DEC-2018
DEFINITION Gateway binary vector pGWB655 DNA, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12218)
AUTHORS Nakamura S, Mano S, Tanaka Y, Ohnishi M, Nakamori C, Araki M, Niwa
T, Nishimura M, Kaminaka H, Nakagawa T, Sato Y, Ishiguro S.
TITLE Gateway binary vectors with the bialaphos resistance gene, bar, as a
selection marker for plant transformation
JOURNAL Biosci. Biotechnol. Biochem. 74 (6), 1315-1319 (2010)
PUBMED 20530878
REFERENCE 2 (bases 1 to 12218)
AUTHORS Tanaka Y, Nakamura S, Ishiguro S, Nakagawa T.
TITLE Direct Submission
JOURNAL Submitted (22-JAN-2010) Contact:Tsuyoshi Nakagawa Shimane
University, Center for Integrated Research in Science; 1060
Nishikawatsu, Matsue, Shimane 690-8504, Japan
REFERENCE 3 (bases 1 to 12218)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 12218)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Biosci.
Biotechnol. Biochem."; date: "2010"; volume: "74"; issue: "6";
pages: "1315-1319"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(22-JAN-2010) Contact:Tsuyoshi Nakagawa Shimane University, Center
for Integrated Research in Science; 1060 Nishikawatsu, Matsue,
Shimane 690-8504, Japan"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT constructed using pPZP221.
FEATURES Location/Qualifiers
source 1..12218
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature complement(54..78)
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
primer_bind 281..297
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 812..1157
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS 1178..1852
/codon_start=1
/label=mRFP1
/note="monomeric derivative of DsRed (Campbell et al.,
2002)"
/translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
TGA"
protein_bind 1862..1986
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
promoter 2023..2053
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 2107..2763
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 3108..3410
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(3454..3578)
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
terminator 3604..3856
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
primer_bind complement(3890..3906)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3914..3930
/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(3938..3968)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(3983..4004)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
terminator complement(4040..4292)
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(4455..5003)
/codon_start=1
/label=BlpR
/note="phosphinothricin acetyltransferase"
/translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
WQLDFSLPVPPRPVLPVTEI"
promoter complement(5055..5234)
/label=NOS promoter
/note="nopaline synthase promoter"
misc_feature complement(5740..5764)
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS 6285..7073
/codon_start=1
/label=SmR
/note="aminoglycoside adenylyltransferase (Murphy, 1985)"
/translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
rep_origin 7322..7910
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(8096..8236)
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(8580..8774)
/direction=LEFT
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
CDS complement(8843..9913)
/codon_start=1
/label=pVS1 RepA
/note="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/translation="VSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
LVMRYRNLIEGEASAGS"
CDS complement(10345..10971)
/codon_start=1
/label=pVS1 StaA
/note="stability protein from the Pseudomonas plasmid pVS1
(Heeb et al., 2000)"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"