pCAG-T7pol vector (V001259)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V001259	pCAG-T7pol	In stock, instant shipping	Buy one, get one free!

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

Basic Vector Information

The pCAG-T7pol is an expression vector designed specifically for the T7 RNA polymerase gene.

Vector Name:: pCAG-T7pol

Antibiotic Resistance:: Ampicillin

Length:: 7457 bp

Type:: Mammalian Expression

Replication origin:: ori

Source/Author:: Ian Wickersham Lab

Copy Number:: High Copy

Promoter:: CAG

5' Primer:: CTCTAGAGCCTCTGCTAACc

3' Primer:: CAGAAGTCAGATGCTCAAG

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

pCAG-T7pol vector Map

Copy Sequence View Map

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

Fuller J, Surtees RA, Slack GS, Mankouri J, Hewson R, Barr JN. Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage. J Virol. 2019 Jul 17;93(15):e00616-19. doi: 10.1128/JVI.00616-19. PMID: 31118258; PMCID: PMC6639264.

pCAG-T7pol vector Sequence

LOCUS       Exported                7457 bp DNA     circular SYN 31-AUG-2024
DEFINITION  Exported.
ACCESSION   V001259
VERSION     .
KEYWORDS    pCAG-T7pol
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7457)
  TITLE     Genetic Neuroengineering Group Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7457)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7457)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7457)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7457
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids,
                     forward primer"
     CDS             1742..4390
                     /codon_start=1
                     /label=T7 RNA polymerase
                     /note="T7 RNA polymerase from Escherichia phage T7.
                     Accession#: P00573"
                     /translation="MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYE
                     MGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQ
                     FLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFK
                     KNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIE
                     STGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGI
                     TGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANV
                     ITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLE
                     FMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYY
                     WLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFE
                     YAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAK
                     KVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVM
                     TLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVE
                     AMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLG
                     QFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHD
                     SFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLN
                     LRDILESDFAFA"
     primer_bind     complement(4479..4498)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4544..4599
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4598..4617)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also
                     called rb-glob-pA-term-R"
     primer_bind     complement(4960..4976)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4984..5000)
                     /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(5008..5038)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5053..5074)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5132..5328
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     polyA_signal    5334..5468
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5536..5553)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5707..6295)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6469..7326)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7327..7431)
                     /label=AmpR promoter

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