pLVX-IRES-tdTomato

pLVX-IRES-tdTomato

载体基本信息

出品公司:	Ybscience
载体名称:	pLVX-IRES-tdTomato
质粒类型:	慢病毒载体；哺乳动物细胞表达载体；双顺反子载体；荧光报告载体
高拷贝/低拷贝:	高拷贝
启动子:	CMV
克隆方法:	多克隆位点，限制性内切酶
载体大小:	8892 bp
5' 测序引物及序列:	CMV-F: CGCAAATGGGCGGTAGGCGTG (Invitrogen)
3' 测序引物及序列:	--
载体标签:	无标签
载体抗性:	氨苄青霉素
筛选标记:	嘌呤霉素(Puromycin)
克隆菌株:	Stbl3
备注:	pLVX-IRES-tdTomato载体含IRES（内部核糖体结合位点）元件； 目的基因和tdTomato荧光蛋白基因以双顺反子形式表达； CMV启动子驱动目的基因的过表达； tdTomato是第三代荧光蛋白，亮度高，适用于流式细胞仪。
稳定性:	稳表达
组成型:	组成型
病毒/非病毒:	慢病毒

 

载体质粒图谱和多克隆位点信息

载体简介

Description
pLVX-IRES-tdTomato is an HIV-1-based, lentiviral expression vector that allows the simultaneous expression of your protein of interest and tdTomato in virtually any mammalian cell type, including primary cells. tdTomato is a member of the family of fruit fluorescent proteins derived from the Discosoma sp. red fluorescent protein, DsRed (1). The vector expresses the two proteins from a bicistronic mRNA transcript, allowing tdTomato to be used as an indicator of transduction efficiency and a marker for selection by flow cytometry.
Expression of the bicistronic transcript is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE) located just upstream of the MCS. An encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), positioned between the MCS and tdTomato, facilitates cap-independent translation of tdTomato from an internal start site at the IRES/tdTomato junction (1).

pLVX-IRES-tdTomato contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral RNA (2), leading to increased viral titers from packaging cells. In addition, the vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RNA out of the nucleus (3). Finally, pLVX-IRES-tdTomato also contains a central polypurine tract/central termination sequence element (cPPT/CTS). During target cell infection, this element creates a central DNA flap that increases nuclear import of the viral genome, resulting in improved vector integration and more efficient transduction (4). The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr) for propagation and selection in bacteria.

Use
pLVX-IRES-tdTomato is designed to constitutively coexpress your protein of interest and tdTomato from PCMV IE when transduced into mammalian cells. Before it can be transduced into target cells, the vector must be packaged into viral particles in HEK293T cells, using our Lenti-X™ HT Packaging System (Cat. Nos. 632160 and 632161). This packaging system allows the safe production of high titer, infectious, replication-incompetent, VSV-G pseudotyped lentiviral particles that can infect a wide range of cell types, including nondividing and primary cells (5).

The presence of tdTomato allows transductants to be visualized by fluorescence microscopy and sorted by flow cytometry with standard FITC filter sets (tdTomato has an excitation maximum of 554 nm and an emission maximum of 581 nm).

载体序列

LOCUS       pLVX-IRES-tdTomato   8892 bp    DNA   circular             2-JUN-2009
COMMENT     Created by Clontech Laboratories Inc.
COMMENT     Cat. No. 631238
COMMENT     This file is created by Vector NTI
            http://www.biofeng.com/
COMMENT     VNTAUTHORNAME|biofeng.com|
FEATURES             Location/Qualifiers
     LTR             1..635
                     /label=5'\LTR
     misc_feature    685..822
                     /label=Packaging\Signal
     misc_binding    1303..1536
                     /label=RRE
     promoter        2185..2787
                     /label=CMV\IE\Promoter
     misc_feature    2842..3416
                     /label=IRES
     CDS             3417..4844
                     /label=tdTomato
     misc_feature    4861..5452
                     /label=WPRE
     LTR             5655..6291
                     /label=3'\LTR
     rep_origin      complement(6760..7433)
                     /label=pUC\ori
     CDS             complement(7578..8574)
                     /label=Amp(R)
     misc_feature    636..653
                     /label=PBS
     misc_feature    2028..2151
                     /label=cPPT/CTS\(DNA\flap)
     misc_feature    2803..2840
                     /label=MCS
BASE COUNT     2285 a      2247 c      2310 g      2050 t 
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 
     2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 
     2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 
     2161 acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa 
     2221 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 
     2281 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 
     2341 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 
     2401 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 
     2461 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 
     2521 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 
     2581 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 
     2641 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 
     2701 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 
     2761 tccatagaag acaccgactc tactagagga tctatttccg gtgaattcct cgagactagt 
     2821 tctagagcgg ccgcggatcc cgcccctctc cctccccccc ccctaacgtt actggccgaa 
     2881 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt 
     2941 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg 
     3001 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc 
     3061 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc 
     3121 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa 
     3181 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc 
     3241 tcacctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg 
     3301 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg 
     3361 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg 
     3421 tgagcaaggg cgaggaggtc atcaaagagt tcatgcgctt caaggtgcgc atggagggct 
     3481 ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca 
     3541 cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc 
     3601 tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg 
     3661 attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg 
     3721 acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg ctgatctaca 
     3781 aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag aagaagacca 
     3841 tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga 
     3901 tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc aagaccatct 
     3961 acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc aagctggaca 
     4021 tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc gagggccgcc 
     4081 accacctgtt cctggggcat ggcaccggca gcaccggcag cggcagctcc ggcaccgcct 
     4141 cctccgagga caacaacatg gccgtcatca aagagttcat gcgcttcaag gtgcgcatgg 
     4201 agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg 
     4261 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg 
     4321 acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca 
     4381 tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact 
     4441 tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga 
     4501 tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga 
     4561 agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg 
     4621 gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga 
     4681 ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc 
     4741 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg 
     4801 gccgccacca cctgttcctg tacggcatgg acgagctgta caagtgaacg cgtctggaac 
     4861 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 
     4921 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 
     4981 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 
     5041 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 
     5101 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 
     5161 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 
     5221 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc 
     5281 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 
     5341 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 
     5401 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tggaattaat 
     5461 tctgcagtcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca 
     5521 atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac 
     5581 ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa 
     5641 aagaaaagag gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc 
     5701 tgtggatcta ccacacacaa ggctacttcc ctgattagca gaactacaca ccagggccag 
     5761 gggtcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagccagata 
     5821 aggtagaaga ggccaataaa ggagagaaca ccagcttgtt acaccctgtg agcctgcatg 
     5881 ggatggatga cccggagaga gaagtgttag agtggaggtt tgacagccgc ctagcatttc 
     5941 atcacgtggc ccgagagctg catccggagt acttcaagaa ctgctgatat cgagcttgct 
     6001 acaagggact ttccgctggg gactttccag ggaggcgtgg cctgggcggg actggggagt 
     6061 ggcgagccct cagatcctgc atataagcag ctgctttttg cctgtactgg gtctctctgg 
     6121 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct 
     6181 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 
     6241 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca 
     6301 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg 
     6361 ccttgacatt gctagcgttt accgtcgacc tctagctaga gcttggcgta atcatggtca 
     6421 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 
     6481 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 
     6541 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 
     6601 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 
     6661 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 
     6721 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 
     6781 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 
     6841 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 
     6901 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 
     6961 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 
     7021 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 
     7081 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 
     7141 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 
     7201 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 
     7261 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 
     7321 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 
     7381 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 
     7441 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 
     7501 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 
     7561 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 
     7621 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 
     7681 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 
     7741 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 
     7801 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 
     7861 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 
     7921 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 
     7981 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 
     8041 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 
     8101 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 
     8161 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 
     8221 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 
     8281 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 
     8341 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 
     8401 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 
     8461 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 
     8521 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga 
     8581 tcgggagatc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 
     8641 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 
     8701 caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa 
     8761 acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac 
     8821 ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac 
     8881 aaacttagta gt