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pGL3 3'UTR reporter WT 1.3 kb CD274 Hs 3'UTR Final vector (V000403)

Price Information

Cat No. Plasmid Name Availability Buy one, get one free! (?)
V000403 pGL3 3'UTR reporter WT 1.3 kb CD274 Hs 3'UTR Final In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
pGL3 3'UTR reporter WT 1.3 kb CD274 Hs 3'UTR Final
Antibiotic Resistance:
Ampicillin
Length:
6166 bp
Type:
Mammalian Expression, Luciferase
Replication origin:
ori
Cloning Method:
Restriction Enzyme
3' Primer:
pGL3-1: GAGCTGACTGGGTTGAAG

pGL3 3'UTR reporter WT 1.3 kb CD274 Hs 3'UTR Final vector Map

Copy Sequence View Map
pGL3 3'UTR reporter WT 1.3 kb CD274 Hs 3'UTR Final6166 bp30060090012001500180021002400270030003300360039004200450048005100540057006000poly(A) signalpause siteSV40 promoterluciferaseWELQut siteL4440oriAmpRAmpR promoterf1 ori

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.

pGL3 3'UTR reporter WT 1.3 kb CD274 Hs 3'UTR Final vector Sequence

LOCUS       40924_22073        6166 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Modified Luciferase expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6166)
  AUTHORS   Coelho MA, de Carne Trecesson S, Rana S, Zecchin D, Moore C, 
            Molina-Arcas M, East P, Spencer-Dene B, Nye E, Barnouin K, Snijders 
            AP, Lai WS, Blackshear PJ, Downward J
  TITLE     Oncogenic RAS Signaling Promotes Tumor Immunoresistance by 
            Stabilizing PD-L1 mRNA.
  JOURNAL   Immunity. 2017 Dec 19;47(6):1083-1099.e6. doi: 
            10.1016/j.immuni.2017.11.016. Epub 2017 Dec 12.
  PUBMED    29246442
REFERENCE   2  (bases 1 to 6166)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6166)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.immuni.2017.11.016"; journalName: "Immunity"; date: 
            "2017-12-19- 19"; volume: "47"; issue: "6"; pages: "1083-1099.e6"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6166
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    7..55
                     /label=poly(A) signal
                     /note="synthetic polyadenylation signal"
     misc_feature    69..160
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from
                     the human alpha-2 globin gene"
     promoter        215..411
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     CDS             447..2096
                     /codon_start=1
                     /label=luciferase
                     /note="firefly luciferase"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     CDS             complement(2526..2537)
                     /codon_start=1
                     /label=WELQut site
                     /note="WELQut protease recognition and cleavage site"
                     /translation="WELQ"
     primer_bind     complement(3665..3682)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3836..4424)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4598..5455)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5456..5560)
                     /label=AmpR promoter
     rep_origin      5587..6042
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"