pCAMBIA1300-35S-mCherry-Golgi vector (Cat. No.: V040635)

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Basic Information

Name:: pCAMBIA1300-35S-mCherry-Golgi

Antibiotic Resistance:: Kanamycin

Length:: 11531 bp

Type:: Protein expression, Subcellular localization

Replication origin:: pVS1

Host:: Plants

Selection Marker:: mCherry

Copy Number:: Low

Promoter:: 35S

Growth Strain(s):: DH5a

Growth Temperature:: 37℃

Expression Method:: Transient

$ 199.0

In stock, 1 week for quality controls

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

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

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.

pCAMBIA1300-35S-mCherry-Golgi vector (Cat. No.: V040635) Sequence

LOCUS       V040635                11531 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V040635
VERSION     V040635
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     promoter        500..845
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
                     /label="CaMV 35S promoter"
     CDS             880..1587
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /transl_table=1
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
                     /label="mCherry"
     terminator      1984..2625
                     /note="terminator and polyadenylation signal from the pea
                     rbcS-E9 gene"
                     /label="E9 terminator"
     primer_bind     complement(2633..2649)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 fwd"
     misc_feature    2852..2876
                     /note="right border repeat from nopaline C58 T-DNA"
                     /label="RB T-DNA repeat"
     CDS             4176..4805
                     /codon_start=1
                     /product="stability protein from the Pseudomonas
                     plasmid pVS1 (Heeb et al., 2000)"
                     /transl_table=1
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI*"
                     /label="pVS1 StaA"
     CDS             5239..6306
                     /codon_start=1
                     /note=""
                     /product="replication protein from the Pseudomonas
                     plasmid pVS1 (Heeb et al., 2000)"
                     /protein_id=""
                     /transl_table=1
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS*"
                     /label="pVS1 RepA"
     rep_origin      6372..6566
                     /note="origin of replication for the Pseudomonas
                     plasmid pVS1 (Heeb et al., 2000)"
                     /label="pVS1 oriV"
     misc_feature    6910..7050
                     /note="basis of mobility region from pBR322"
                     /label="bom"
     rep_origin      complement(7236..7824)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(7911..8705)
                     /codon_start=1
                     /gene="aphA-3"
                     /note="confers resistance to kanamycin"
                     /product="aminoglycoside phosphotransferase"
                     /protein_id=""
                     /transl_table=1
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF*"
                     /label="KanR"
     misc_feature    9130..9154
                     /note="left border repeat from nopaline C58 T-DNA"
                     /label="LB T-DNA repeat"
     polyA_signal    9232..9406
                     /note="cauliflower mosaic virus polyadenylation signal"
                     /label="CaMV poly(A) signal"
     CDS             complement(9446..10471)
                     /codon_start=1
                     /note="confers resistance to hygromycin"
                     /product="hygromycin B phosphotransferase"
                     /transl_table=1
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K*"
                     /label="HygR"
     promoter        complement(10539..11216)
                     /note="cauliflower mosaic virus 35S promoter with a
                     duplicated enhancer region"
                     /label="CaMV 35S promoter (enhanced)"
     protein_bind    11407..11428
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     promoter        11443..11473
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    11481..11497
                     /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-β-D-thiogalactopyranoside (IPTG)."
                     /label="lac operator"
     primer_bind     11505..11521
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"