pLVTHM慢病毒载体使用说明

pLVTHM

pLVTHM 载体基本信息：

载体名称:

pLVTHM 质粒类型: 哺乳动物细胞慢病毒表达载体, RNAi 载体， Cre/loxP 载体

高拷贝/低拷贝: 高拷贝

启动子:

EF-1 alpha 克隆方法: 多克隆位点，限制性内切酶

载体大小:

11085 bp 5' 测序引物及序列:

EF-1a Forward: TCAAGCCTCAGACAGTGGTTC 3' 测序引物及序列:

EGFP-N: CGTCGCCGTCCAGCTCGACCAG 载体标签:

C-EGFP 载体抗性: 氨苄

备注: 载体使用Stbl3感受态细胞扩增质粒，可以减少载体基因

重组几率。pLVTHM 载体用来替代pLVTH ，允许shRNA 定

向 克隆入载体。

稳定性:

/ 组成型:

-- 病毒/非病毒: 慢病毒

pLVTHM 载体质粒图谱和多克隆位点信息：

pLVTHM载体简介：

pLVTHM allows for direct cloning of shRNAs into the lentiviral vectors and replaces the old pLVTH system from Trono lab.

pLVTHM is similar to pLVTH, but contains a 3bp substitution that generates a unique MluI site for direct cloning of an shRNA into MluI-ClaI.

Please note that there is an additional ClaI site in this vector that is blocked by Dam methylation (this site does not appear in the depositor's full sequence). The plasmid needs to be grown in a Dam+ bacterial strain in order to use ClaI for cloning.

Note that ClaI has lower salt concentration requirements than MluI. One way to handle this is to digest with ClaI for 45 minutes, followed by addition of diluted MluI buffer and MluI, then incubation for 90 more minutes. Be sure not to use a large amount of vector (1.7 ug to 2.5 ug is known to work).

Also, if your shRNA is already in pSUPER (or another plasmid under the control of the PolIII promoter) you may use the EcoRI-ClaI sites to replace the H1 promoter in pLVTHM with the

H1-shRNA cassette from pSUPER (or another plasmid).

pLVTHM载体序列：

ORIGIN

1 TTGGAAGGGC TAATTCACTC CCAAAGAAGA CAAGATATCC TTGATCTGTG GATCTACCAC

61 ACACAAGGCT ACTTCCCTGA TTAGCAGAAC TACACACCAG GGCCAGGGGT CAGATATCCA 121 CTGACCTTTG GATGGTGCTA CAAGCTAGTA CCAGTTGAGC CAGATAAGGT AGAAGAGGCC 181 AATAAAGGAG AGAACACCAG CTTGTTACAC CCTGTGAGCC TGCATGGGAT GGATGACCCG 241 GAGAGAGAAG TGTTAGAGTG GAGGTTTGAC AGCCGCCTAG CATTTCATCA CGTGGCCCGA 301 GAGCTGCATC CGGAGTACTT CAAGAACTGC TGATATCGAG CTTGCTACAA GGGACTTTCC 361 GCTGGGGACT TTCCAGGGAG GCGTGGCCTG GGCGGGACTG GGGAGTGGCG AGCCCTCAGA 421 TCCTGCATAT AAGCAGCTGC TTTTTGCCTG TACTGGGTCT CTCTGGTTAG ACCAGATCTG

481 AGCCTGGGAG CTCTCTGGCT AACTAGGGAA CCCACTGCTT AAGCCTCAAT AAAGCTTGCC 541 TTGAGTGCTT CAAGTAGTGT GTGCCCGTCT GTTGTGTGAC TCTGGTAACT AGAGATCCCT 601 CAGACCCTTT TAGTCAGTGT GGAAAATCTC TAGCAGTGGC GCCCGAACAG GGACTTGAAA 661 GCGAAAGGGA AACCAGAGGA GCTCTCTCGA CGCAGGACTC GGCTTGCTGA AGCGCGCACG 721 GCAAGAGGCG AGGGGCGGCG ACTGGTGAGT ACGCCAAAAA TTTTGACTAG CGGAGGCTAG 781 AAGGAGAGAG ATGGGTGCGA GAGCGTCAGT ATTAAGCGGG GGAGAATTAG ATCGCGATGG 841 GAAAAAATTC GGTTAAGGCC AGGGGGAAAG AAAAAATATA AATTAAAACA TATAGTATGG 901 GCAAGCAGGG AGCTAGAACG ATTCGCAGTT AATCCTGGCC TGTTAGAAAC ATCAGAAGGC 961 TGTAGACAAA TACTGGGACA GCTACAACCA TCCCTTCAGA CAGGATCAGA AGAACTTAGA 1021 TCATTATATA ATACAGTAGC AACCCTCTAT TGTGTGCATC AAAGGATAGA GATAAAAGAC 1081 ACCAAGGAAG CTTTAGACAA GATAGAGGAA GAGCAAAACA AAAGTAAGAC CACCGCACAG 1141 CAAGCGGCCG CTGATCTTCA GACCTGGAGG AGGAGATATG AGGGACAATT GGAGAAGTGA 1201 ATTATATAAA TATAAAGTAG TAAAAATTGA ACCATTAGGA GTAGCACCCA CCAAGGCAAA 1261 GAGAAGAGTG GTGCAGAGAG AAAAAAGAGC AGTGGGAATA GGAGCTTTGT TCCTTGGGTT 1321 CTTGGGAGCA GCAGGAAGCA CTATGGGCGC AGCGTCAATG ACGCTGACGG TACAGGCCAG 1381 ACAATTATTG TCTGGTATAG TGCAGCAGCA GAACAATTTG CTGAGGGCTA TTGAGGCGCA 1441 ACAGCATCTG TTGCAACTCA CAGTCTGGGG CATCAAGCAG CTCCAGGCAA GAATCCTGGC 1501 TGTGGAAAGA TACCTAAAGG ATCAACAGCT CCTGGGGATT TGGGGTTGCT CTGGAAAACT 1561 CATTTGCACC ACTGCTGTGC CTTGGAATGC TAGTTGGAGT AATAAATCTC TGGAACAGAT 1621 TTGGAATCAC ACGACCTGGA TGGAGTGGGA CAGAGAAATT AACAATTACA CAAGCTTAAT 1681 ACACTCCTTA ATTGAAGAAT CGCAAAACCA GCAAGAAAAG AATGAACAAG AATTATTGGA 1741 ATTAGATAAA TGGGCAAGTT TGTGGAATTG GTTTAACATA ACAAATTGGC TGTGGTATAT 1801 AAAATTATTC ATAATGATAG TAGGAGGCTT GGTAGGTTTA AGAATAGTTT TTGCTGTACT 1861 TTCTATAGTG AATAGAGTTA GGCAGGGATA TTCACCATTA TCGTTTCAGA CCCACCTCCC 1921 AACCCCGAGG GGACCCGACA GGCCCGAAGG AATAGAAGAA GAAGGTGGAG AGAGAGACAG 1981 AGACAGATCC ATTCGATTAG TGAACGGATC TCGACGGTAT CGCGATGTCG ACGATAAGCT 2041 TTGCAAAGAT GGATAAAGTT TTAAACAGAG AGGAATCTTT GCAGCTAATG GACCTTCTAG 2101 GTCTTGAAAG GAGTGGGAAT TGGCTCCGGT GCCCGTCAGT GGGCAGAGCG CACATCGCCC 2161 ACAGTCCCCG AGAAGTTGGG GGGAGGGGTC GGCAATTGAA CCGGTGCCTA GAGAAGGTGG 2221 CGCGGGGTAA ACTGGGAAAG TGATGTCGTG TACTGGCTCC GCCTTTTTCC CGAGGGTGGG 2281 GGAGAACCGT ATATAAGTGC AGTAGTCGCC GTGAACGTTC TTTTTCGCAA CGGGTTTGCC 2341 GCCAGAACAC AGGTAAGTGC CGTGTGTGGT TCCCGCGGGC CTGGCCTCTT TACGGGTTAT 2401 GGCCCTTGCG TGCCTTGAAT TACTTCCACT GGCTGCAGTA CGTGATTCTT GATCCCGAGC 2461 TTCGGGTTGG AAGTGGGTGG GAGAGTTCGA GGCCTTGCGC TTAAGGAGCC CCTTCGCCTC