pLenti-Bi-cistronic
pLenti-Bi-cistronic
编号 | 载体名称 |
北京华越洋VECT75612 | pLenti-Bi-cistronic |
pLenti-Bi-cistronic载体基本信息：
启动子: | RSV ,PGK |
复制子: | M13 ori,ori ,SV40 ori |
终止子: | SV40 poly(A) signal |
质粒分类: | 病毒系列，慢病毒双表达载体 |
质粒大小: | 8292bp |
原核抗性: | Kan |
筛选标记: | Puro |
克隆菌株: | Stbl3 |
培养条件: | 37℃，有氧 LB |
表达宿主: | 哺乳细胞 |
诱导方式: | 无须诱导，瞬时表达 |
5'测序引物: | 根据序列设计引物 |
3'测序引物: | 根据序列设计引物 |
pLenti-Bi-cistronic载体图谱：
pLenti-Bi-cistronic载体简介：
This study was aimed to construct, package and purify the recombinant lentivirus vector carrying the firefly luciferase gene (FLUC) and red fluorescent protein gene (RFP) and to transfect the recombinant lentivirus into HeLa cells, so as to observe the expression levels of these two genes. The FLUC and RFP genes were amplified by RT-PCR and inserted in the lentiviral expression vector (pLenti-Bi-cistronic) to construct the lentiviral vector pLenti-FLUC-RFP. The viral particles were generated by cotransfection of 293T cells with pLenti-FLUC-RFP and three packaging vectors, and the virus titer was determined by calculating the percentage of RFP positive cells. After transfection of pLenti-FLUC-RFP into HeLa cells, the expression of RFP was observed by fluorescent microscopy, and the activity of FLUC was determined by luciferase reporter gene assay kit. The results showed that the inserting orientation of the RFP and FLUC genes in the lentiviral vector pLenti-FLUC-RFP were verified by restriction analysis. Targeted RFP and FLUC sequences were confirmed by DNA sequencing. The final titer obtained was 1×10TU/ml. The expressions of RFP and FLUC were observed in the transfected HeLa cells. It is concluded that the pLenti-III-FLUC-RFP recombinant lentivirus vector carrying RFP gene and FLUC gene with high viral titer is constructed and packaged successfully, and provides experimental basis for studying dynamic distribution of mesenchymal stem cells in vivo.
pLenti-Bi-cistronic载体序列：
ORIGIN
1 ttttggattg aagccaatat gataatgagg gggtggagtt tgtgacgtgg cgcggggcgt
61 gggaacgggg cgggtgacgt agtagtgtgg cggaagtgtg atgttgcaag tgtggcggaa
121 cacatgtaag cgacggatgt ggcaaaagtg acgtttttgg tgtgcgccgg tgtacacagg
181 aagtgacaat tttcgcgcgg ttttaggcgg atgttgtagt aaatttgggc gtaaccgagt
241 aagatttggc cattttcgcg ggaaaactga ataagaggaa gtgaaatctg aataattttg
301 tgttactcat agcgcgtaat acggcagacc tcagcgctag attattgaag catttatcag
361 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
421 gttccgcgca catttccccg aaaagtgcca cctgacgtta actataacgg tcctaaggta
481 gcgaaaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
541 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
601 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
661 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct
721 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
781 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
841 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
901 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
961 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
1021 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
1081 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
1141 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
1201 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
1261 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
1321 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
1381 aaagtaagac caccgcacag caagcccgct gatcttcaga cctggaggag gagatatgag
1441 ggacattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
1501 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
1561 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
1621 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
1681 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
1741 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
1801 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
1861 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
1921 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
1981 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
2041 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
2101 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
2161 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa
2221 ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg acggtatcga
2281 aagcttggga ttcgaattta aaagaaaagg ggggattggg gggtacagtg caggggaaag
2341 aatagtagac ataatagcaa cagacataca aactaaagaa ctacaaaaac aaattacaaa
2401 aattcaaaat tttcgggttt ttcgaaccta gcagacgtcg gcagtgaaaa aaatgcttta
2461 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
2521 ttaacaacaa gaattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt
2581 tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatctc acctaggctc
2641 gagggatccc ccgggcgatc tgacggttca ctaaacgagc tctgcttata taggcctccc
2701 accgtacacg ccacctcgac ataaattcta ccgggtaggg gaggcgcttt tcccaaggca
2761 gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg
2821 gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc
2881 cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc
2941 gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag
3001 caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc
3061 cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg
3121 gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca
3181 aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctatcg
3241 atcaattgag tacttacgta ggtaccgaat tcgatatcgg gcccgcggcc gcgtctagat
3301 gagtcgagta cccatacgac gtcccagact acgcttgagt ttaaacacgc gtggtgtgga
3361 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
3421 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
3481 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
3541 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
3601 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggccatgac
3661 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccctcggg ccgtacgcac
3721 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtggacc cggaccgcca
3781 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
3841 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
3901 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
3961 gctggccgcg cagcaacaga tggaagggct cctggcgccg caccggccca aggagcccgc
4021 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
4081 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
4141 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
4201 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg
4261 ttccggaaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
4321 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
4381 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
4441 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
4501 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
4561 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
4621 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg
4681 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
4741 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
4801 gcgtctcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgtc
4861 cggatggaag ggctaattca ctcccaacga atacaagatc tgctttttgc ttgtactggg
4921 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
4981 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
5041 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
5101 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
5161 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
5221 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
5281 aatgtatctt atcatgtctg gcatctatgt cgggtgcgga gaaagaggta atgaaatggc
5341 attatgggta ttatgggtct gcattaatga atcggccaac gatcccggtg tgaaataccg
5401 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
5461 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
5521 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
5581 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
5641 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
5701 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
5761 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
5821 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
5881 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
5941 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
6001 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
6061 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
6121 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
6181 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
6241 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
6301 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
6361 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
6421 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
6481 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
6541 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
6601 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
6661 gttaatagtt tgcgcaacgt tgttgaaaaa ggatcttcac ctagatcctt ttcacgtaga
6721 aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga
6781 caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat
6841 agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct
6901 ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttctcgccg ccaaggatct
6961 gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg
7021 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
7081 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
7141 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
7201 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
7261 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
7321 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
7381 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
7441 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
7501 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
7561 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
7621 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
7681 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
7741 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
7801 tcttctgaat tttgttaaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
7861 atcggcaaca tcccttataa atcaaaagaa tagaccgcga tagggttgag tgttgttcca
7921 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
7981 gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttgcggtcg
8041 aggtgccgta aagctctaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
8101 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
8161 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgcgcg cttaatgcgc
8221 cgctacaggg cgcgtccatt cgccattcag gatcgaatta attcttaatt aacatcatca
8281 ataatatacc tt
//
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