pCMV- Tet3G

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  • Model: PVT11010
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pCMV-Tet3G

PVT11010  2ug

 

pCMV-Tet3G Description

pCMV-Tet3G is a Mammal plasmid.

 

pCMV-Tet3G Informaiton

Promoter: CMV

Replicon: pUC ori, SV40 ori

Terminator: SV40 poly (A) signal

Plasmid classification: mammalian cells, tetracycline regulatory system vectors

Plasmid size: 7139bp

Prokaryotic resistance: Amp

Clonal strain: DH5 alpha

Culture conditions: 37 C, aerobic LB

Expression host: lactation cells

Induction: tetracycline induction

5'sequencing primers: CMV-F:CGCAAATGGGCGGTAGGCGTG

Primers for 3'sequencing: primers designed based on sequences

 

pCMV-Tet3G Introduction

pCMV-Tet3G Vector expresses Tet-On® 3G, a tetracycline-controlled transactivator that exhibits high activity in the presence of the inducer doxycycline (Dox) and exceptionally low activity in its absence. Tet-On 3G results from the fusion of amino acids 1–207 of a mutant Tet repressor (TetR) to 39 amino acids that form three minimal "F"-type transcriptional activation domains from the herpes simplex virus VP16 protein. Tet-On 3G was derived from Tet-On Advanced (1–4); as a result, it’s fully synthetic, lacks cryptic splice sites, and is codon-optimized for stable expression in mammalian cells. Compared to both of its predecessors, this 3rd generation Tet-On transactivator demonstrates increased sensitivity to Dox (1). Constitutive expression of Tet-On 3G is driven by the human cytomegalovirus immediately early promoter (PCMV IE). Note: An EF1α version of this vector is available for cell lines in which the CMV promoter is silenced. pCMV-Tet3G is used to develop stable Tet-On 3G cell lines, which are hosts for Tet-inducible gene expression systems. To create a Tet-inducible expression system, a vector containing a gene of interest under the control of the Tet-inducible TRE3G promoter (PTRE3G) is transfected into a Tet-On 3G cell line. The addition of Dox to the system causes Tet-On 3G to undergo a conformational change that allows it to bind to PTRE3G, activating transcription of the gene of interest in a highly dose-dependent manner.

 

pCMV-Tet3G Multiple cloning site

pCMV-Tet3G-vector

 

 

 

pCMV-Tet3G Sequence

LOCUS       Exported                7139 bp ds-DNA     circular SYN 05-SEP-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled 6
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7139)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Monday, September 5, 2016 from SnapGene Viewer 3.1.4
           
FEATURES             Location/Qualifiers
     source          1..7139
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        89..468
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        469..672
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             775..1521
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /note="Tet-On(R) 3G"
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    1531..1665
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2385..2973)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3144..4004)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4005..4109)
                     /gene="bla"
                     /note="AmpR promoter"
     polyA_signal    4222..4356
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     CDS             complement(4781..4801)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     intron          4931..4996
                     /note="small t intron"
                     /note="simian virus 40 (SV40) small t antigen intron"
     CDS             complement(5416..6210)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(6571..6900)
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      6585..6720
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
ORIGIN
        1 ctcgaggagc ttggcccatt gcatacgttg tatccatatc ataatatgta catttatatt
       61 ggctcatgtc caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa
      121 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      661 ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt
      721 gacctccata gaagacaccg ggaccgatcc agcctccgcg gccccgaatt caccatgtct
      781 agactggaca agagcaaagt cataaactct gctctggaat tactcaatgg agtcggtatc
      841 gaaggcctga cgacaaggaa actcgctcaa aagctgggag ttgagcagcc taccctgtac
      901 tggcacgtga agaacaagcg ggccctgctc gatgccctgc caatcgagat gctggacagg
      961 catcataccc actcctgccc cctggaaggc gagtcatggc aagactttct gcggaacaac
     1021 gccaagtcat accgctgtgc tctcctctca catcgcgacg gggctaaagt gcatctcggc
     1081 acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag
     1141 caaggcttct ccctggagaa cgcactgtac gctctgtccg ccgtgggcca ctttacactg
     1201 ggctgcgtat tggaggaaca ggagcatcaa gtagcaaaag aggaaagaga gacacctacc
     1261 accgattcta tgcccccact tctgaaacaa gcaattgagc tgttcgaccg gcagggagcc
     1321 gaacctgcct tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag
     1381 tgcgaaagcg gcgggccgac cgacgccctt gacgattttg acttagacat gctcccagcc
     1441 gatgcccttg acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttga
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