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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