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pNEU-hMbPylRS-4xU6M15 vector (V017142)

Price Information

Cat No. Plasmid Name Availability Add to cart
V017142 pNEU-hMbPylRS-4xU6M15 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

Vector Name:
pNEU-hMbPylRS-4xU6M15
Antibiotic Resistance:
Ampicillin
Length:
7948 bp
Type:
Protein expression
Replication origin:
ori
Host:
Mammalian cells
Selection Marker:
Neo/G418
Promoter:
U6
Growth Strain(s):
DH5a
Growth Temperature:
37℃

pNEU-hMbPylRS-4xU6M15 vector Map

Copy Sequence View Map
pNEU-hMbPylRS-4xU6M157948 bp30060090012001500180021002400270030003300360039004200450048005100540057006000630066006900720075007800U6 promoterCMV enhancerCMV promoterT7 promoterPyrrolysine--tRNA ligasebGH poly(A) signalf1 oriSV40 promoterNeoR/KanRSV40 poly(A) signalM13 revlac operatorlac promoterCAP binding siteoriAmpRAmpR 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.

pNEU-hMbPylRS-4xU6M15 vector Sequence

LOCUS       V017142                 7948 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V017142
VERSION     V017142
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 7948)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1138..1378)
                     /label="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     enhancer        1617..1996
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1997..2200
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        2245..2263
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2289..3545
                     /gene="pylS"
                     /label="Pyrrolysine--tRNA ligase"
                     /note="Pyrrolysine--tRNA ligase from Methanosarcina
                     barkeri. Accession#: Q6WRH6"
     polyA_signal    3581..3805
                     /label="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3851..4279
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4293..4622
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     CDS             4689..5480
                     /label="NeoR/KanR"
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    5657..5790
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5825..5841)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(5849..5865)
                     /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(5873..5903)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5918..5939)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6227..6815)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(6989..7846)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(join(7847..7948,1..3))
                     /label="AmpR promoter"