JD633 vector (V012098)

Price Information

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V012098 JD633 In stock, 1 week for quality controls

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Vector Name:
JD633
Antibiotic Resistance:
Kanamycin
Length:
21667 bp
Type:
Binary vector
Replication origin:
ori
Selection Marker:
Hygromycin ; PvUbi2:hpt II
Promoter:
Ubi

JD633 vector Map

JD63321667 bp100020003000400050006000700080009000100001100012000130001400015000160001700018000190002000021000pVS1 StaApVS1 RepApVS1 oriVpGEX 3'bomL4440oriKanRLB T-DNA repeatCaMV poly(A) signalHygRCAP binding sitelac promoterlac operatorM13 revattB1attB23xHANOS terminatorUbi promoterCas9nucleoplasmin NLSHSP terminatorM13 fwdT7 promoterT3 promoterM13 revlac operatorlac promoterCAP binding sitegRNA scaffoldM13 fwdRB T-DNA repeat

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.

JD633 vector Sequence

Copy Sequence

Download GenBank File(.gb)

LOCUS       40924_1424       21667 bp DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR-Cas9 vector including GRF4-GIF1. For wheat and other monocots
            CRISPR experiments. Includes two AarI sites for gRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 21667)
  AUTHORS   Debernardi JM, Tricoli DM, Ercoli MF, Hayta S, Ronald P, Palatnik 
            JF, Dubcovsky J
  TITLE     A GRF-GIF chimeric protein improves the regeneration efficiency of 
            transgenic plants.
  JOURNAL   Nat Biotechnol. 2020 Oct 12. pii: 10.1038/s41587-020-0703-0. doi: 
            10.1038/s41587-020-0703-0.
  PUBMED    33046875
REFERENCE   2  (bases 1 to 21667)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 21667)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2020 Oct 12. pii: 10.1038/s41587-020-0703-0. doi: 
            10.1038/s41587-020-0703-0."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..21667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1233..1859
                     /codon_start=1
                     /label=pVS1 StaA
                     /note="stability protein from the Pseudomonas plasmid pVS1
                     (Heeb et al., 2000)"
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2291..3361
                     /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      3430..3624
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(3860..3882)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3968..4108
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4123..4140)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4294..4882)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4972..5763)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    6188..6212
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    complement(6290..6464)
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(6508..7530)
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     protein_bind    9470..9491
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        9506..9536
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9544..9560
                     /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)."
     primer_bind     9568..9584
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    11629..11653
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      11650..11659
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(13571..13591)
                     /label=attB2
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             13621..13710
                     /codon_start=1
                     /label=3xHA
                     /note="three tandem HA epitope tags"
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      13744..13996
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        14011..16007
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             16022..20125
                     /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             20126..20173
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      20184..20432
                     /label=HSP terminator
                     /note="efficient transcription terminator from the
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     primer_bind     20980..20996
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        21006..21024
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(21072..21090)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(21111..21127)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    21135..21151
                     /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(21159..21189)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(21204..21225)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_RNA        21264..21339
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(21356..21372)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    21575..21599
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"