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pSG5 Large T vector (V006716) Gene synthesis in pSG5 Large T backbone

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V006716 pSG5 Large T In stock, instant shipping

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

Basic Vector Information

Vector Name:
pSG5 Large T
Antibiotic Resistance:
Ampicillin
Length:
6252 bp
Type:
Mammalian Expression
Replication origin:
ori
Copy Number:
High Copy
Cloning Method:
Restriction Enzyme
5' Primer:
T7
3' Primer:
SV40pA-R
Growth Strain(s):
DH10B

pSG5 Large T vector Map

Copy Sequence View Map
pSG5 Large T6252 bp30060090012001500180021002400270030003300360039004200450048005100540057006000SV40 promoterbeta-globin intronpCAG-FT7 promoterlarge T antigenSV40 poly(A) signalL4440oriAmpRAmpR promoterpBRforEcopGEX 3'pRS-markerf1 oriM13 fwd

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.

pSG5 Large T vector Sequence

LOCUS       40924_39907        6252 bp DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6252)
  TITLE     T antigen
REFERENCE   2  (bases 1 to 6252)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6252)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..358
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     intron          389..961
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     primer_bind     969..988
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids,
                     forward primer"
     promoter        1016..1034
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1076..3199
                     /codon_start=1
                     /label=large T antigen
                     /note="large T antigen from SV40 (simian virus 40)"
                     /translation="MDKVLNREESLQLMDLLGLERSAWGNIPLMRKAYLKKCKEFHPDK
                     GGDEEKMKKMNTLYKKMEDGVKYAHQPDFGGFWDATEIPTYGTDEWEQWWNAFNEENLF
                     CSEEMPSSDDEATADSQHSTPPKKKRKVEDPKDFPSELLSFLSHAVFSNRTLACFAIYT
                     TKEKAALLYKKIMEKYSVTFISRHNSYNHNILFFLTPHRHRVSAINNYAQKLCTFSFLI
                     CKGVNKEYLMYSALTRDPFSVIEESLPGGLKEHDFNPEEAEETKQVSWKLVTEYAMETK
                     CDDVLLLLGMYLEFQYSFEMCLKCIKKEQPSHYKYHEKHYANAAIFADSKNQKTICQQA
                     VDTVLAKKRVDSLQLTREQMLTNRFNDLLDRMDIMFGSTGSADIEEWMAGVAWLHCLLP
                     KMDSVVYDFLKCMVYNIPKKRYWLFKGPIDSGKTTLAAALLELCGGKALNVNLPLDRLN
                     FELGVAIDQFLVVFEDVKGTGGESRDLPSGQGINNLDNLRDYLDGSVKVNLEKKHLNKR
                     TQIFPPGIVTMNEYSVPKTLQARFVKQIDFRPKDYLKHCLERSEFLLEKRIIQSGIALL
                     LMLIWYRPVAEFAQSIQSRIVEWKERLDKEFSLSVYQKMKFNVAMGIGVLDWLRNSDDD
                     DEDSQENADKNEDGGEKNMEDSGHETGIDSQSQGSFQAPQSSQSVHDHNQPYHICRGFT
                     CFKKPPTPPPEPET"
     polyA_signal    3253..3374
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3408..3425)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3579..4167)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4341..5198)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5199..5303)
                     /label=AmpR promoter
     primer_bind     5371..5389
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     primer_bind     complement(5427..5449)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5549..5568
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     rep_origin      complement(5635..6090)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     6231..6247
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"