GmScreamM8-pFLEV vector (Cat. No.: V009937)

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Basic Information

Note: GmScreamM8-pFLEV is a ~5.3 kb plant binary/cloning vector built around the GmScreamM8 promoter—a constitutive, high-activity soybean eEF1A-derived promoter (~1.6 kb)[1] that drives EGFP as reporter, terminated by the NOS terminator. Backbone carries pUC ori, AmpR, and a lac operator for bacterial repression. GenBank: KX252734[2]

Name:: GmScreamM8-pFLEV

Antibiotic Resistance:: Ampicillin

Length:: 5261 bp

Type:: Cloning vector

Replication origin:: ori

Source/Author:: Zhang N, McHale LK, Finer JJ.

Growth Temperature:: 37℃

$ 386.8

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.

References

[1]Zhang N, McHale LK, Finer JJ. Isolation and characterization of "GmScream" promoters that regulate highly expressing soybean (Glycine max Merr.) genes. Plant Science. 2015;241:189-198

[2]Hernandez-Garcia CM, Bouchard RA, Rushton PJ, et al. High level transgenic expression of soybean (Glycine max) GmERF and Gmubi gene promoters isolated by a novel promoter analysis pipeline. BMC Plant Biology. 2010;10(1):237

GmScreamM8-pFLEV vector (Cat. No.: V009937) Sequence

LOCUS       40924_1174        5261 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector GmScreamM8-pFLEV, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5261)
  AUTHORS   Zhang N, McHale LK, Finer JJ.
  TITLE     Isolation and characterization of 'GmScream' promoters that regulate
            highly expressing soybean (Glycine max Merr.) genes
  JOURNAL   Plant Sci. 241, 189-198 (2015)
  PUBMED    26706070
REFERENCE   2  (bases 1 to 5261)
  AUTHORS   Zhang N.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-MAY-2016) Horticulture and Crop Science, Ohio State 
            University, 1680 Madison Ave, Wooster, OH 44691, USA
REFERENCE   3  (bases 1 to 5261)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5261)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant Sci. 
            241, 189-198 (2015)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (17-MAY-2016) Horticulture and Crop Science, Ohio State University, 
            1680 Madison Ave, Wooster, OH 44691, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5261
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      405..1978
                     /label=GmScreamM8 promoter
                     /note="GmScreamM8 promoter"
                     /regulatory_class="promoter"
     CDS             1981..2697
                     /label=EGFP
                     /note="enhanced GFP"
     terminator      2733..2985
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(3040..3056)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3064..3080)
                     /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)."
     promoter        complement(3088..3118)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3133..3154)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3442..4030)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4204..5061)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5062..5166)
                     /label=AmpR promoter