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GW luc-basic vector (V009927)

Price Information

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V009927 GW luc-basic 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

GW luc basic was designed for the highthroughput cloning of deletion mutant.

Vector Name:
GW luc-basic
Antibiotic Resistance:
Ampicillin
Length:
6531 bp
Type:
Cloning vector
Replication origin:
ori
Source/Author:
Del Vecchio I, Zuccotti A, Canneva F, Lenzken SC, Racchi M.

GW luc-basic vector Map

Copy Sequence View Map
GW luc-basic6531 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300ColE1-derived plasmid replication originmultiple cloning site 1attR1lac UV5 promoterCmRccdBattR2multiple cloning site 2luciferaseSV40 poly(A) signalmultiple cloning site 3oriAmpRAmpR promoterf1 oripoly(A) signalpause site

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

  • Del Vecchio I, Zuccotti A, Canneva F, Lenzken SC, Racchi M. Development of the first Gateway firefly luciferase vector and use of reverse transcriptase in FLOE (Fluorescently Labeled Oligonucleotide Extension) reactions [published correction appears in Plasmid.2011 Sep;66(3):186]. Plasmid. 2007;58(3):269-274. doi:10.1016/j.plasmid.2007.07.002

GW luc-basic vector Sequence

LOCUS       40924_1254        6531 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector GWluc-basic, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6531)
  AUTHORS   Del Vecchio I, Zuccotti A, Canneva F, Lenzken SC, Racchi M.
  TITLE     Development of the first Gateway firefly luciferase vector and use 
            of reverse transcriptase in FLOE (Fluorescently Labeled 
            Oligonucleotide Extension) reactions
  JOURNAL   Plasmid 58 (3), 269-274 (2007)
  PUBMED    17707908
REFERENCE   2  (bases 1 to 6531)
  AUTHORS   Del Vecchio I, Zuccotti A, Canneva F, Lanni C, Lenzken C, Govoni S, 
            Racchi M.
  TITLE     Improved firefly luciferase vector to rapidly recombine promoter 
            regions
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 6531)
  AUTHORS   Del Vecchio I, Zuccotti A, Canneva F, Lanni C, Lenzken C, Govoni S, 
            Racchi M.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-FEB-2007) Centre of Excellence in Applied Biology - 
            Experimental and Applied Pharmacology Dept., University of Pavia, 
            Via Taramelli 14, Pavia 27100, Italy
REFERENCE   4  (bases 1 to 6531)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6531)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plasmid"; 
            date: "2007"; volume: "58"; issue: "3"; pages: "269-274"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (22-FEB-2007) Centre of Excellence in Applied Biology - Experimental
            and Applied Pharmacology Dept., University of Pavia, Via Taramelli 
            14, Pavia 27100, Italy"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6531
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..28
                     /label=multiple cloning site 1
                     /note="multiple cloning site 1"
     protein_bind    33..157
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        182..212
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             266..922
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             1267..1569
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(1613..1737)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    1742..1770
                     /label=multiple cloning site 2
                     /note="multiple cloning site 2"
     CDS             1801..3450
                     /codon_start=1
                     /label=luciferase
                     /note="firefly luciferase"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     polyA_signal    complement(3494..3615)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    3717..3728
                     /label=multiple cloning site 3
                     /note="multiple cloning site 3"
     rep_origin      4031
                     /label=ColE1-derived plasmid replication origin
                     /note="ColE1-derived plasmid replication origin"
     rep_origin      complement(4034..4622)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4796..5653)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5654..5758)
                     /label=AmpR promoter
     rep_origin      5785..6240
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     polyA_signal    6371..6419
                     /label=poly(A) signal
                     /note="synthetic polyadenylation signal"
     misc_feature    6433..6524
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from
                     the human alpha-2 globin gene"