pHDE-35S-Cas9-mCherry-UBQ vector (V000409)

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V000409	pHDE-35S-Cas9-mCherry-UBQ	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

The pHDE-35S-Cas9-mCherry-UBQ is for CRISPR/Cas9-mediated gene editing in Arabidopsis, it offers a visual screening method to identify Cas9-free plants and facilitates the generation of two guide RNAs (gRNAs).

Vector Name:: pHDE-35S-Cas9-mCherry-UBQ

Antibiotic Resistance:: Spectinomycin

Length:: 16084 bp

Type:: Plant Expression, CRISPR

Replication origin:: ori

Host:: Plants

Selection Marker:: Hygromycin ; mCherry fluorescence

Promoter:: CaMV 35S

Cloning Method:: Gibson Cloning

5' Primer:: CTGTGGTTGGCATGCACATAC

Growth Strain(s):: stbl3

pHDE-35S-Cas9-mCherry-UBQ vector Map

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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

Gao X, Chen J, Dai X, Zhang D, Zhao Y. An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing. Plant Physiol. 2016;171(3):1794-1800. doi:10.1104/pp.16.00663

pHDE-35S-Cas9-mCherry-UBQ vector Sequence

LOCUS       40924_24398       16084 bp DNA     circular SYN 13-MAY-2021
DEFINITION  For CRISPR/Cas9 mediated gene editing in Arabidopsis; provides a 
            visual screen for Cas9-free plants. Enable production of two gRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16084)
  AUTHORS   Gao X, Chen J, Dai X, Zhang D, Zhao Y
  TITLE     An effective strategy for reliably isolating heritable and Cas9-free
            Arabidopsis mutants generated by CRISPR/Cas9-mediated genome 
            editing.
  JOURNAL   Plant Physiol. 2016 May 15. pii: pp.00663.2016.
  PUBMED    27208253
REFERENCE   2  (bases 1 to 16084)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 16084)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Physiol. 2016 May 15. pii: pp.00663.2016."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..16084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    complement(1..25)
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             508..1194
                     /codon_start=1
                     /label=mCherry
                     /note="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /translation="DNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLK
                     VTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVT
                     VTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRL
                     KLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTG
                     GMDELYK"
     terminator      1203..1455
                     /label=MAS terminator
                     /note="mannopine synthase terminator"
     promoter        2600..2783
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     CDS             2835..3845
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDIGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRP"
     terminator      3898..4150
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        4283..4628
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             4633..8736
                     /codon_start=1
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             8749..8769
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(9281..9298)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21)
                     Forward"
     primer_bind     complement(9281..9297)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9290..9312)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    complement(9519..9543)
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             10071..10859
                     /codon_start=1
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      11106..11694
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11848..11865
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    complement(11880..12020)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     12106..12128
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      complement(12364..12558)
                     /direction=LEFT
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(12627..13691)
                     /codon_start=1
                     /label=pVS1 RepA
                     /note="replication protein from the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     CDS             complement(14128..14754)
                     /codon_start=1
                     /label=pVS1 StaA
                     /note="stability protein from the Pseudomonas plasmid pVS1
                     (Heeb et al., 2000)"
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"