pCAMBIA2201 vector (V008746)

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

Basic Vector Information

Vector Name:
pCAMBIA2201
Antibiotic Resistance:
Chloramphenicol
Length:
11785 bp
Type:
Binary vector
Replication origin:
ori
Host:
Plants
Source/Author:
Hajdukiewicz P, Svab Z, Maliga P.
Selection Marker:
Neomycin/G418(Geneticin)
Copy Number:
High copy number
Promoter:
CaMV 35S (enhanced)
5' Primer:
M13 fwd
3' Primer:
M13 rev

pCAMBIA2201 vector Map

pCAMBIA220111785 bp50010001500200025003000350040004500500055006000650070007500800085009000950010000105001100011500synthetic first gus exon (modular)GUS6xHisNOS terminatorRB T-DNA repeatpVS1 StaApVS1 RepApVS1 oriVbomoriCmRLB T-DNA repeatCaMV poly(A) signalNeoR/KanRCaMV 35S promoter (enhanced)CAP binding sitelac promoterlac operatorM13 revMCSM13 fwdCaMV 35S promoter

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.

pCAMBIA2201 vector Sequence

LOCUS       40924_8801       11785 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Binary vector pCAMBIA-2201, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11785)
  AUTHORS   Hajdukiewicz P, Svab Z, Maliga P.
  TITLE     The small, versatile pPZP family of Agrobacterium binary vectors for
            plant transformation
  JOURNAL   Plant Mol. Biol. 25 (6), 989-994 (1994)
  PUBMED    7919218
REFERENCE   2  (bases 1 to 11785)
  AUTHORS   Roberts C, Rajagopal S, Smith LM, Nguyen TA, Yang W, Nugrohu S, Ravi
            KS, Vijayachandra K, Harcourt RL, Dransfield L, Desamero N, Slamet 
            I, Hadjukiewicz P, Svab Z, Maliga P, Mayer JE, Keese PK, Kilian A, 
            Jefferson RA.
  TITLE     A comprehensive set of modular vectors for advanced manipulations 
            and efficient transformation of plants
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 11785)
  AUTHORS   Roberts C, Rajagopal S, Smith LM, Nguyen TA, Yang W, Nugrohu S, Ravi
            KS, Vijayachandra K, Harcourt RL, Dransfield L, Desamero N, Slamet 
            I, Hadjukiewicz P, Svab Z, Maliga P, Mayer JE, Keese PK, Kilian A, 
            Jefferson RA.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-FEB-2000) CAMBIA, Clunies Ross St, Black Mountain / 
            GPO Box 3200, Canberra, ACT 2601, Australia
REFERENCE   4  (bases 1 to 11785)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 11785)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant Mol. 
            Biol."; date: "1994"; volume: "25"; issue: "6"; pages: "989-994"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (15-FEB-2000) CAMBIA, Clunies Ross St, Black Mountain / GPO Box 
            3200, Canberra, ACT 2601, Australia"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..11785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     exon            2..16
                     /note="synthetic first gus exon (modular)"
     intron          17..206
                     /label=cat1 intron
                     /note="castor bean catalase intron, modified"
     CDS             201..2024
                     /label=GUS
                     /note="beta-glucuronidase"
     CDS             2031..2048
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      2083..2335
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    2357..2381
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             3681..4307
                     /label=pVS1 StaA
                     /note="stability protein from the Pseudomonas plasmid pVS1
                     (Heeb et al., 2000)"
     CDS             4744..5808
                     /label=pVS1 RepA
                     /note="replication protein from the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     rep_origin      5877..6071
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     misc_feature    6415..6555
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(6741..7329)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7603..8259)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     misc_feature    8787..8811
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    complement(8889..9063)
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(9123..9911)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     promoter        complement(9980..10657)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a
                     duplicated enhancer region"
     protein_bind    10848..10869
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        10884..10914
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10922..10938
                     /label=lac operator
                     /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     10946..10962
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    10972..11028
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(11032..11048)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        11425..11770
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"