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pLVX-tdTomato-N1载体质粒基本信息
pLVX-tdTomato-N1质粒图谱载体图谱和pLVX-tdTomato-N1载体序列质粒序列多克隆位点信息
pLVX-tdTomato-N1质粒载体简介
pLVX-tdTomato-N1载体描述
pLVX-tdTomato-N1 is an HIV-1-based, lentiviral expression vector that allows you to express your gene of interest fused to tdTomato, a member of the family of fruit fluorescent proteins (1) derived from the Discosoma sp. red fluorescent protein, DsRed (2). Genes cloned into the multiple cloning site (MCS), located upstream of the tdTomato coding sequence, are expressed as N-terminal tdTomato fusion proteins. Expression of the fusion protein is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE), located just upstream of the MCS. Lentiviral particles derived from the vector allow the expression of tdTomato fusion proteins in virtually any cell type, including primary cells. The unmodified vector expresses tdTomato, and may be used to produce marker virus to optimize infection protocols.
pLVX-tdTomato-N1 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 and transgene RNA (3), leading to increased viral titers from packaging cells, and enhanced expression of your gene of interest in target 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 (4). Finally, pLVX-tdTomato-N1 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 (5).
In addition to lentiviral elements, pLVX-tdTomato-N1 contains a puromycin resistance gene (Puror) under the control of the murine phosphoglycerate kinase (PGK) promoter (PPGK) for the selection of stable transductants. The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr) for propagation and selection in bacteria.
To construct a fusion protein, the gene of interest must be cloned into pLVX-tdTomato-N1 so that it is in-frame with the tdTomato coding sequence. The inserted sequence should include an initiation codon (ATG) and lack in-frame stop codons.The fusion protein is constitutively expressed when pLVX-tdTomato-N1 is transduced into target cells. Before the vector can be transduced, however, it must be transfected into 293T packaging cells with our Lenti-X HT Packaging System (Cat. Nos. 632160 and 632161). This packaging system allows you to safely produce high titer, infectious, replication-incompetent, VSV-G pseudotyped lentiviral particles that can infect a wide range of cell types, including non-dividing and primary cells (6).
Selection of Stable Transfectants
Selectable marker: plasmid confers resistance to puromycin.
Propagation in E. coli
Suitable host strains: DH5α, DH10B and other general purpose strains.
Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Copy number: high
Excitation and emission maxima of tdTomato
Excitation maximum = 554 nm
Emission maximum = 581 nm
pLVX-tdTomato-N1质粒序列载体序列
LOCUS pLVX-tdTomato-N1 9498 bp DNA circular 7-OCT-2009
COMMENT Created by Clontech Laboratories Inc.
COMMENT Cat. No. 632563
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_feature 5466..6057
/label=WPRE
CDS complement(8184..9180)
/label=Amp(R)
misc_binding 1303..1536
/label=RRE
misc_binding 636..653
/label=PBS
LTR 6260..6896
/label=3'\LTR
promoter 4324..4832
/label=PGK\Promoter
CDS 4853..5452
/label=Puro(R)
CDS 2875..4305
/label=tdTomato
misc_feature 2028..2151
/label=cPPT/CTS\(DNA\flap)
promoter 2185..2787
/label=CMV\IE\Promoter
rep_origin complement(7366..8039)
/label=pUC\ori
misc_feature 2815..2867
/label=MCS
BASE COUNT 2330 a 2523 c 2546 g 2099 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 tcgctagcgc taccggactc agatctcgag
2821 ctcaagcttc gaattctgca gtcgacggta ccgcgggccc gggatccacc ggtcatggtg
2881 agcaagggcg aggaggtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc
2941 atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
3001 cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg
3061 tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat
3121 tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
3181 ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag
3241 gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg
3301 ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc
3361 caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa gaccatctac
3421 atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc
3481 acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac
3541 cacctgttcc tggggcatgg caccggcagc accggcagcg gcagctccgg caccgcctcc
3601 tccgaggaca acaacatggc cgtcatcaaa gagttcatgc gcttcaaggt gcgcatggag
3661 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
3721 ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac
3781 atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc
3841 cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
3901 gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc
3961 tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag
4021 accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc
4081 gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc
4141 atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg
4201 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc
4261 cgccaccacc tgttcctgta cggcatggac gagctgtaca agtgagcggc cgcgactcta
4321 gataattcta ccgggtaggg gaggcgcttt tcccaaggca gtctggagca tgcgctttag
4381 cagccccgct gggcacttgg cgctacacaa gtggcctctg gcctcgcaca cattccacat
4441 ccaccggtag gcgccaaccg gctccgttct ttggtggccc cttcgcgcca ccttctactc
4501 ctcccctagt caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa
4561 tggaagtagc acgtctcact agtctcgtgc agatggacag caccgctgag caatggaagc
4621 gggtaggcct ttggggcagc ggccaatagc agctttgctc cttcgctttc tgggctcaga
4681 ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg
4741 cccgaaggtc ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc
4801 tgttctcctc ttcctcatct ccgggccttt cgacctgcag cccaagctta ccatgaccga
4861 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
4921 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
4981 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
5041 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
5101 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
5161 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
5221 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
5281 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
5341 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
5401 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaccgcgtct
5461 ggaacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
5521 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
5581 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
5641 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
5701 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
5761 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
5821 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc
5881 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
5941 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
6001 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctggaa
6061 ttaattctgc agtcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc
6121 taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt
6181 cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
6241 tttaaaagaa aagaggggac tggaagggct aattcactcc caacgaagac aagatatcct
6301 tgatctgtgg atctaccaca cacaaggcta cttccctgat tagcagaact acacaccagg
6361 gccaggggtc agatatccac tgacctttgg atggtgctac aagctagtac cagttgagcc
6421 agataaggta gaagaggcca ataaaggaga gaacaccagc ttgttacacc ctgtgagcct
6481 gcatgggatg gatgacccgg agagagaagt gttagagtgg aggtttgaca gccgcctagc
6541 atttcatcac gtggcccgag agctgcatcc ggagtacttc aagaactgct gatatcgagc
6601 ttgctacaag ggactttccg ctggggactt tccagggagg cgtggcctgg gcgggactgg
6661 ggagtggcga gccctcagat cctgcatata agcagctgct ttttgcctgt actgggtctc
6721 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
6781 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
6841 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta
6901 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
6961 agaggccttg acattgctag cgttttaccg tcgacctcta gctagagctt ggcgtaatca
7021 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
7081 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
7141 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
7201 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
7261 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
7321 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
7381 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
7441 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
7501 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
7561 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
7621 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
7681 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
7741 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
7801 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
7861 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
7921 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
7981 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
8041 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
8101 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
8161 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
8221 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
8281 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
8341 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
8401 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
8461 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
8521 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
8581 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
8641 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
8701 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
8761 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
8821 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
8881 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
8941 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
9001 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
9061 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
9121 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
9181 gacggatcgg gagatcaact tgtttattgc agcttataat ggttacaaat aaagcaatag
9241 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
9301 actcatcaat gtatcttatc atgtctggat caactggata actcaagcta accaaaatca
9361 tcccaaactt cccaccccat accctattac cactgccaat tacctgtggt ttcatttact
9421 ctaaacctgt gattcctctg aattattttc attttaaaga aattgtattt gttaaatatg
9481 tactacaaac ttagtagt
//
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pLVX-tdTomato-N1质粒载体应用举例
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