pDsRed-Express-C1载体

pDsRed-Express-C1载体描述pDsRed-Express-C1 is a mammalian expression vector that encodes DsRed-Express, a variant of Discosoma sp. red fluorescent protein (DsRed; 1). DsRed-Express contains nine amino acid substitutions, which improve the solubility of the protein and reduce the time from transfection to detection of red fluorescence (2). In addition, these substitutions reduce the level of residual green emission (2). When DsRed-Express is expressed in mammalian cell cultures, redemitting cells can be detected by either fluorescence microscopy or flow cytometry 8–12 hours after transfection (DsRed-Express excitation and emission maxima = 557 nm and 579 nm, respectively). Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed- Express displays a reduced tendency to aggregate (2). The DsRed-Express coding sequence is human codon-optimized for high expression in mammalian cells (3). The multiple cloning site (MCS) in pDsRed-Express-C1 is positioned between the DsRed-Express coding sequence and the SV40 polyadenylation signal (SV40 poly A). Genes cloned into the MCS will be expressed as fusions to the C-terminus of DsRed-Express if they are in the same reading frame as DsRed-Express and there are no intervening stop codons. A Kozak consensus translation initiation site upstream of DsRed-Express increases the translation efficiency in eukaryotic cells (4). SV40 poly A signals downstream of the MCS direct proper processing of the 3' end of mRNA transcripts. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin resistance cassette (Neor)—consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene—allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli.pDsRed-Express-C1 can be used to construct fusions to the C-terminus of DsRed-Express. If a fusion construct retains the fluorescent properties of the native DsRed-Express protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene should be cloned into pDsRed-Express-C1 so that it is in frame with the DsRed-Express coding sequences, with no intervening in-frame stop codons. The recombinant DsRed-Express vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (5). pDsRed-Express-C1 can also be used as a cotransfection marker; the unmodified vector will express DsRed-Express.pDsRed-Express-C1载体含有以下元件： Human cytomegalovirus (CMV) immediate early promoter: 1–589Enhancer region: 59–465;TATA box: 554–560Transcription start point: 583C→G mutation to remove Sac I site: 569 Discosoma sp. human codon-optimized Red Fluorescent Protein (DsRed-Express) geneKozak consensus translation initiation site: 606–616Start codon (ATG): 613–615; Stop codon: 1357–1359CGC→GCC (Arg-2 to Ala) mutation: 616–618AAG→GAG (Lys-5 to Glu) mutation: 625–627AAC→GAC (Asn-6 to Asp) mutation: 628–630ACC→TCC (Thr-21 to Ser) mutation: 673–675CAC→ACC (His-41 to Thr) mutation: 733–735AAC→CAG (Asn-42 to Gln) mutation: 736–738GTG→GCC (Val-44 to Ala) mutation: 742–744TGC→TCC (Cys-117 to Ser) mutation: 961–963ACC→GCC (Thr-217 to Ala) mutation: 1261–1263Last amino acid in DsRed-Express: 1285–1287 MCS: 1288–1344 SV40 early mRNA polyadenylation signalPolyadenylation signals: 1498–1503 & 1527–1532; mRNA3' ends: 1536 & 1648 f1 single-strand DNA origin: 1595–2050 (Packages the noncoding strand of DsRed-Express.) Bacterial promoter for expression of Kanr gene–35 region: 2112–2117; –10 region: 2135–2140Transcription start point: 2147 SV40 origin of replication: 2391–2526 SV40 early promoterEnhancer (72-bp tandem repeats): 2224–2295 & 2296–236721-bp repeats: 2371–2991, 2392–2412 & 2414–2431Early promoter element: 2447–2453Major transcription start points: 2443, 2481, 2487 & 2492 Kanamycin/neomycin resistance geneNeomycin phosphotransferase coding sequences:Start codon (ATG): 2576–2578; stop codon: 3368–3370G→A mutation to remove Pst I site: 2758C→A (Arg to Ser) mutation to remove BssH II site: 3104 Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signalPolyadenylation signals: 3606–3611 & 3619–3624 pUC plasmid replication origin: 3955–4598Propagation in E. coli Suitable host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA productionrequires a host containing an F plasmid such as JM109 or XL1-Blue. Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts. E. coli replication origin: pUC Copy number: ≈500 Plasmid incompatibility group: pMB1/Col E1Excitation and emission maxima of DsRed-Express Excitation maximum = 557 nm Emission maximum = 579 nm

LOCUS       pDsRed-Express-C1	4680 bp 	DNA	SYNDEFINITION  pDsRed-Express-C1ACCESSION   KEYWORDS    SOURCE        ORGANISM  other sequences; artificial sequences; vectors.FEATURES             Location/Qualifiers     source          1..4680                     /organism="pDsRed-Express-C1"                     /mol_type="other DNA"     promoter        10..562                     /label="CMV_immearly_promoter"     misc_feature    65..352                     /label="CAG_enhancer"     misc_feature    519..539                     /label="CMV_fwd_primer"     promoter        520..589                     /label="CMV_promoter"     CDS             complement(598..1377)                     /label="ORF frame 1"                     /translation="MADYDQLSRSGGSRARGTVDCRIRSLSSRSQEQVVAALGALVLL                     HDGVVLVVGGDVQLGVHVVVAGQLHGLLGHVDGLELHQVVAAVLQLQGLVDLALQHAV                     AGVQALGGGLPAHSLLLHYGAVGGEVHADELHLVDEGAVLQGGVLGHGHHAAVLEVHH                     ALPLEALGEGQLLVVGDVGGVLHVHLGAVLELGGQDVPGEGQGAALGHLQLGGLGALV                     GAALALALDLELVAVHGALHAHLEAHELLDDVLGGGHGGDR*"     CDS             613..1359                     /label="ORF frame 1"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     gene            616..1287                     /label="DsRed_Express"                     /gene="DsRed_Express"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     misc_feature    complement(793..813)                     /label="dsRed1_N_primer"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     misc_feature    1205..1228                     /label="dsRed1_C_primer"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     terminator      1361..1589                     /label="SV40_PA_terminator"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     misc_feature    1559..1578                     /label="EBV_rev_primer"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     rep_origin      complement(1728..2034)                     /label="f1_origin"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     promoter        2113..2141                     /label="AmpR_promoter"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     misc_feature    complement(2207..2227)                     /label="pBABE_3_primer"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     misc_feature    complement(2213..2428)                     /label="SV40_enhancer"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     promoter        2225..2493                     /label="SV40_promoter"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     rep_origin      2392..2469                     /label="SV40_origin"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     misc_feature    2454..2473                     /label="SV40pro_F_primer"                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDG                     GVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGE                     IHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAE                     GRHHLFLRSRAQASNSAVDGTAGPGSTGSR*"     CDS             2576..3370                     /label="ORF frame 2"                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE                     EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"     gene            2579..3367                     /label="NeoR/KanR"                     /gene="NeoR/KanR"                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE                     EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"     CDS             complement(2885..3421)                     /label="ORF frame 3"                     /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA                     VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA                     VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ                     PPHCISHDGYFLGRSKVR*"     terminator      3545..3814                     /label="TK_PA_terminator"                     /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA                     VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA                     VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ                     PPHCISHDGYFLGRSKVR*"     rep_origin      3962..4581                     /label="pBR322_origin"                     /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA                     VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA                     VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ                     PPHCISHDGYFLGRSKVR*"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 CCGGTCGCCA CCATGGCCTC CTCCGAGGAC GTCATCAAGG AGTTCATGCG CTTCAAGGTG  661 CGCATGGAGG GCTCCGTGAA CGGCCACGAG TTCGAGATCG AGGGCGAGGG CGAGGGCCGC  721 CCCTACGAGG GCACCCAGAC CGCCAAGCTG AAGGTGACCA AGGGCGGCCC CCTGCCCTTC  781 GCCTGGGACA TCCTGTCCCC CCAGTTCCAG TACGGCTCCA AGGTGTACGT GAAGCACCCC  841 GCCGACATCC CCGACTACAA GAAGCTGTCC TTCCCCGAGG GCTTCAAGTG GGAGCGCGTG  901 ATGAACTTCG AGGACGGCGG CGTGGTGACC GTGACCCAGG ACTCCTCCCT GCAGGACGGC  961 TCCTTCATCT ACAAGGTGAA GTTCATCGGC GTGAACTTCC CCTCCGACGG CCCCGTAATG 1021 CAGAAGAAGA CTATGGGCTG GGAGGCCTCC ACCGAGCGCC TGTACCCCCG CGACGGCGTG 1081 CTGAAGGGCG AGATCCACAA GGCCCTGAAG CTGAAGGACG GCGGCCACTA CCTGGTGGAG 1141 TTCAAGTCCA TCTACATGGC CAAGAAGCCC GTGCAGCTGC CCGGCTACTA CTACGTGGAC 1201 TCCAAGCTGG ACATCACCTC CCACAACGAG GACTACACCA TCGTGGAGCA GTACGAGCGC 1261 GCCGAGGGCC GCCACCACCT GTTCCTGAGA TCTCGAGCTC AAGCTTCGAA TTCTGCAGTC