Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000360 | pHEE401E | 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:
- pHEE401E
- Antibiotic Resistance:
- Kanamycin
- Length:
- 17112 bp
- Type:
- Plant Expression, CRISPR ; Plant binary vector
- Replication origin:
- ori
- Selection Marker:
- Hygromycin
- Copy Number:
- High Copy
- Promoter:
- CaMV35S(enhanced)
- 3' Primer:
- M13-rev
pHEE401E vector 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.
pHEE401E vector Sequence
LOCUS 40924_24423 17112 bp DNA circular SYN 13-MAY-2021
DEFINITION Egg cell-specific promoter-controlled expression of
3×FLAG-NLS-zCas9-NLS. Contains gRNA scaffold for insertion of target
sequence (U6-26 promoter), Hyg resistance.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 17112)
AUTHORS Wang ZP, Xing HL, Dong L, Zhang HY, Han CY, Wang XC, Chen QJ
TITLE Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently
generates homozygous mutants for multiple target genes in
Arabidopsis in a single generation.
JOURNAL Genome Biol. 2015 Jul 21;16:144. doi: 10.1186/s13059-015-0715-0.
PUBMED 26193878
REFERENCE 2 (bases 1 to 17112)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 17112)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Genome
Biol."; date: "2015-07-21"; pages: "
10.1186/s13059-015-0715-0"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..17112
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 78..100
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 92..109
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 93..109
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 119..542
/label=AtU6-26
/note="Arabidopsis U6-26 gene promoter"
CDS 822..1610
/codon_start=1
/label=SmR
/note="aminoglycoside adenylyltransferase (Murphy, 1985)"
/translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
misc_RNA 1765..1840
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
CDS 3410..3475
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 3482..3502
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3527..7627
/codon_start=1
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 7628..7675
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
terminator 7685..8317
/label=E9 terminator
/note="terminator and polyadenylation signal from the pea
rbcS-E9 gene"
primer_bind complement(8332..8348)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8332..8348)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8345..8367)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 8356..8372
/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(8380..8410)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(8425..8446)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 8637..9313
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
CDS 9380..10402
/codon_start=1
/label=HygR
/note="aminoglycoside phosphotransferase from E. coli"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
K"
polyA_signal 10446..10620
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
misc_feature complement(10698..10722)
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS 11147..11938
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
rep_origin 12028..12616
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 12770..12787
/label=L4440
/note="L4440 vector, forward primer"
misc_feature complement(12802..12942)
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 13028..13050
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
rep_origin complement(13286..13480)
/direction=LEFT
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
CDS complement(13549..14619)
/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(15051..15677)
/codon_start=1
/label=pVS1 StaA
/note="stability protein from the Pseudomonas plasmid pVS1
(Heeb et al., 2000)"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
misc_feature complement(16978..17002)
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"