pKillerRed- Mem

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  • Model: PVT10851
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pKillerRed-Mem


Catalog No. PVT10851
Packing 2ug

 

pKillerRed-Mem Information

Function Mammal reporter plasmid

Promoter: CMV promoter

Replicator: Ori, F1 ori, SV40 ori

Terminator: SV40 poly (A) signal

Plasmid classification: lactation cell plasmids; lactating reporter plasmid; fluorescent localization plasmid

Plasmid size: 4787bp

Plasmid label: C-mem, C-KillerRed

Prokaryotic resistance: kanamycin Kan

Screening markers: neomycin Neo/G418

Cloned strain: Escherichia coli DH5 alpha

Culture conditions: 37 centigrade, aerobic, LB

Expression host: mammalian cells such as 293T

 

pKillerRed-Mem Description

PKillerRed-mem is a plasmid which is red fluorescence located in cell membrane of mammalian cells.

pKillerRed-mem is a mammalian expression vector encoding membrane-targeted KillerRed. KillerRed localized on cellular membrane can be used for effective light-induced cell killing.KillerRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al. 1996]. Membrane localization signal (MLS) of neuromodulin is linked to the KillerRed N-terminus. The MLS (Nterminal 20 amino acid residues of neuromodulin) contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets KillerRed to cellular membranes [Skene and Virág 1989]. pKillerRed-mem vector can be used as a source of MLS-KillerRed hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A) direct proper processing of the 3’-end of the reporter mRNA. SV40 early promoter (PSV40) provides neomycin resistance gene (Neor ) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr ) in E. coli. Kanr /Neor gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.pKillerRed-mem vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of memrane-targeted KillerRed in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman 1985].Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 µg/ml) to E. coli hosts. Copy number in E. coli is about 500.

 

pKillerRed-Mem Sequence

LOCUS      Exported                4787 bp ds-DNA     circular SYN 17-AUG-2017
DEFINITION  synthetic circular DNA
KEYWORDS    pKillerRed-mem.dna
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4787)
FEATURES             Location/Qualifiers
     source          1..4787
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    591..671
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    679..738
                     /label=mem
     misc_feature    739..1452
                     /label=KillerRed
     polyA_signal    1575..1696
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1703..2158)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2185..2289
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2291..2648
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2499..2634
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2683..3477
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3709..3756
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4085..4673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccaccgg tcgccaccat gctgtgctgt atgagaagaa ccaaacaggt tgaaaagaat
      721 gatgaggacc aaaagatctc cgagggcggc cccgccctgt tccagagcga catgaccttc
      781 aaaatcttca tcgacggcga ggtgaacggc cagaagttca ccatcgtggc cgacggcagc
      841 agcaagttcc cccacggcga cttcaacgtg cacgccgtgt gcgagaccgg caagctgccc
      901 atgagctgga agcccatctg ccacctgatc cagtacggcg agcccttctt cgcccgctac
      961 cccgacggca tcagccattt cgcccaggag tgcttccccg agggcctgag catcgaccgc
     1021 accgtgcgct tcgagaacga cggcaccatg accagccacc acacctacga gctggacgac
     1081 acctgcgtgg tgagccgcat caccgtgaac tgcgacggct tccagcccga cggccccatc
     1141 atgcgcgacc agctggtgga catcctgccc aacgagaccc acatgttccc ccacggcccc
     1201 aacgccgtgc gccagctggc cttcatcggc ttcaccaccg ccgacggcgg cctgatgatg
     1261 ggccacttcg acagcaagat gaccttcaac ggcagccgcg ccatcgagat ccccggccca
     1321 cacttcgtga ccatcatcac caagcagatg agggacacca gcgacaagcg cgaccacgtg
     1381 tgccagcgcg aggtggccta cgcccacagc gtgccccgca tcaccagcgc catcggtagc
     1441 gacgaggatt aaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg
     1501 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     1561 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     1621 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     1681 tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta
     1741 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     1801 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     1861 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     1921 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     1981 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     2041 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     2101 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca
     2161 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
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