Price Information
Cat No. | Plasmid Name | Availability | Add to cart |
---|---|---|---|
V010863 | pEarleyGate 103 | 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:
- pEarleyGate 103
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 12411 bp
- Type:
- Plant Vectors
- Replication origin:
- ori
- Source/Author:
- Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS.
- Copy Number:
- High copy number
- Promoter:
- MAS
pEarleyGate 103 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.
pEarleyGate 103 vector Sequence
LOCUS 40924_16475 12411 bp DNA circular SYN 01-JAN-1980 DEFINITION Gateway(R)-compatible plant transformation vector with GFP and 6xHis C-terminal tags. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12411) AUTHORS Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS. TITLE Gateway-compatible vectors for plant functional genomics and proteomics. JOURNAL Plant J. 2006;45:616-29. PUBMED 16441352 REFERENCE 2 (bases 1 to 12411) AUTHORS Pikaard Lab TITLE Direct Submission REFERENCE 3 (bases 1 to 12411) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Plant J."; date: "2006"; volume: "45"; pages: "616-29" COMMENT SGRef: number: 2; type: "Journal Article" COMMENT The sequence was corrected by inserting a G at position 8519. FEATURES Location/Qualifiers source 1..12411 /mol_type="other DNA" /organism="synthetic DNA construct" misc_feature 1..25 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" terminator complement(111..363) /label=MAS terminator /note="mannopine synthase terminator" CDS complement(376..924) /codon_start=1 /label=BlpR /note="phosphinothricin acetyltransferase" /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF WQLDFSLPVPPRPVLPVTEI" promoter complement(930..1310) /label=MAS promoter /note="mannopine synthase promoter (Velten et al., 1984)" promoter 2306..2651 /label=CaMV 35S promoter /note="strong constitutive promoter from cauliflower mosaic virus" protein_bind 2663..2782 /label=attR1 /note="recombination site for the Gateway(R) LR reaction" promoter 2812..2842 /label=lac UV5 promoter /note="E. coli lac promoter with an 'up' mutation" CDS 2896..3552 /codon_start=1 /label=CmR /note="chloramphenicol acetyltransferase" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" CDS 3897..4199 /codon_start=1 /label=ccdB /note="CcdB, a bacterial toxin that poisons DNA gyrase" /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" protein_bind complement(4243..4367) /label=attR2 /note="recombination site for the Gateway(R) LR reaction" CDS 4390..5100 /codon_start=1 /label=mgfp5 /note="GFP with folding enhancement mutations" /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN FKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITHGMDELYK" CDS 5107..5124 /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" terminator 5182..5889 /label=OCS terminator /note="octopine synthase terminator" misc_feature 6133..6157 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" CDS 7457..8083 /codon_start=1 /label=pVS1 StaA /note="stability protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI" CDS 8515..9585 /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" rep_origin 9654..9848 /label=pVS1 oriV /note="origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" misc_feature 10192..10332 /label=bom /note="basis of mobility region from pBR322" rep_origin complement(10518..11106) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(11196..11987) /codon_start=1 /label=KanR /note="aminoglycoside phosphotransferase" /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"