pEarleyGate 301 vector (V010856)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V010856	pEarleyGate 301	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

pEarleyGate 301 is a Gateway-compatible promoterless plant transformation vector with a C-terminal HA tag.

Vector Name:: pEarleyGate 301

Antibiotic Resistance:: Chloramphenicol

Length:: 10379 bp

Type:: Plant Vectors

Replication origin:: ori

Source/Author:: Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS.

Selection Marker:: Bar

Copy Number:: High copy number

Promoter:: MAS

Growth Strain(s):: DB3.1

Growth Temperature:: 37℃

pEarleyGate 301 vector Map

Copy Sequence View 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

Romera-Branchat M, Severing E, Pocard C, Ohr H, Vincent C, Née G, Martinez-Gallegos R, Jang S, Andrés F, Madrigal P, Coupland G. Functional Divergence of the Arabidopsis Florigen-Interacting bZIP Transcription Factors FD and FDP. Cell Rep. 2020 Jun 2;31(9):107717.

pEarleyGate 301 vector Sequence

LOCUS       40924_16510       10379 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Gateway(R)-compatible promoterless plant transformation vector with 
            a C-terminal HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10379)
  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 10379)
  AUTHORS   Pikaard Lab
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 10379)
  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 6487.
FEATURES             Location/Qualifiers
     source          1..10379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    3..27
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     terminator      complement(113..365)
                     /label=MAS terminator
                     /note="mannopine synthase terminator"
     CDS             complement(378..926)
                     /codon_start=1
                     /label=BlpR
                     /note="phosphinothricin acetyltransferase"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     promoter        complement(932..1312)
                     /label=MAS promoter
                     /note="mannopine synthase promoter (Velten et al., 1984)"
     protein_bind    1364..1483
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1513..1543
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1597..2253
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2598..2900
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(2944..3068)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3070..3096
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     terminator      3150..3857
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     misc_feature    4101..4125
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             5425..6051
                     /codon_start=1
                     /label=pVS1 StaA
                     /note="stability protein from the Pseudomonas plasmid pVS1
                     (Heeb et al., 2000)"
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             6483..7553
                     /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      7622..7816
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     misc_feature    8160..8300
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(8486..9074)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9164..9955)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"