| 出品公司: | Clontech |
|---|---|
| 载体名称: | pIRESneo3, pIRESneo 3 |
| 质粒类型: | 哺乳动物细胞表达载体 |
| 启动子: | CMV |
| 表达水平: | 高 |
| 克隆方法: | 多克隆位点，限制性内切酶 |
| 载体大小: | 5260bp |
| 5' 测序引物: | CMV-F |
| 5' 测序引物序列: | 5'-CGCAAATGGGCGGTAGGCGTG-3' |
| 3' 测序引物: | pIRESrv |
| 3' 测序引物序列: | 5'-GCCCTAGATGCATGCTCG-3' |
| 载体标签: | 无 |
| 载体抗性: | 氨苄青霉素（Ampicillin） |
| 筛选标记: | 新霉素（Neomycin） |
| 克隆菌株: | DH5α, HB101 |
| 宿主细胞（系）: | 常规细胞系，293、CV-1、CHO等 |
| 备注: |
pIRESneo3 contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA.
|
| 产品目录号: | 631621 |
| 稳定性: | 稳表达 或 瞬表达 |
| 组成型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
买家导航
pIRESneo 3载体基本信息
pIRESneo 3载体图谱质粒图谱和多克隆位点信息
pIRESneo 3载体简介
Description:
pIRESneo3 contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA (1–3). After selection with G418, nearly all surviving colonies will stably express the gene of interest, thus decreasing the need to screen large numbers of colonies to find functional clones. To select for cells that express high levels of the gene of interest, the selective pressure for antibiotic resistance was increased by shifting the neomycin phosphotransferase gene downstream to a less optimal position for translation as directed by the IRES sequence (1). By decreasing the level of expression of the antibiotic resistance marker, the selective pressure on the entire expression cassette is increased, resulting in the selection of cells that express the entire transcript, including the gene of interest, at high levels.
The expression cassette of pIRESneo3 contains the human cytomegalovirus (CMV) major immediate early promoter/enhancer followed by a multiple cloning site (MCS) that precedes stop codons in all three reading frames, a synthetic intron known to enhance the stability of the mRNA (4), the ECMV IRES followed by the neomycin phosphotransferase (NPT II) gene, and the polyadenylation signal from SV40. Ribosomes can enter the bicistronic mRNA at the 5' end to translate the gene of interest and at the ECMV IRES to translate the antibiotic resistance marker.
Use:
When using the pIRESneo3 Vector, the antibiotic exerts selective pressure on the entire expression cassette; thus, a high dose of antibiotic will select for cells expressing a high level of the gene of interest. This selective pressure also ensures that the expression of the gene of interest will be stable over time in culture. Unless your expression experiments require a pure population of cells, you can use the pool of cells surviving selection instead of isolating and characterizing clonal cell lines. We recommend selecting mammalian cultures in 500–1,300 μg/ml G418 (Cat. No. 631307) depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of G418 to determine optimal selection concentration.
pIRESneo 3载体序列
LOCUS pIRESneo3 5260 bp DNA circular SYN DEFINITION pIRESneo3 ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors. COMMENT This file is created by Vector NTI http://www.biofeng.com/ COMMENT ORIGDB|GenBank| COMMENT VNTAUTHORNAME|biofeng.com| FEATURES Location/Qualifiers source 1..5260 /organism="pIRESneo3" /mol_type="other DNA" promoter 236..852 /label="CMV_immearly_promoter" misc_feature 315..602 /label="CAG_enhancer" misc_feature 769..789 /label="CMV_fwd_primer" promoter 863..881 /label="T7_promoter" misc_feature 1015..1310 /label="synth_int" misc_feature 1337..1900 /label="IRES" CDS 1821..2750 /label="ORF frame 3" gene 1947..2747 /label="NeoR/KanR" /gene="NeoR/KanR" CDS complement(2265..2789) /label="ORF frame 3" terminator 2773..3001 /label="SV40_PA_terminator" misc_feature 2971..2990 /label="EBV_rev_primer" misc_feature 3024..3043 /label="EBV_rev_primer" promoter complement(3099..3117) /label="M13_reverse_primer" misc_feature complement(3116..3138) /label="M13_pUC_rev_primer" promoter complement(3152..3181) /label="lac_promoter" rep_origin complement(3490..4109) /label="pBR322_origin" gene complement(4264..5124) /label="Ampicillin" /gene="Ampicillin" CDS complement(4264..5124) /label="ORF frame 2" promoter complement(5166..5194) /label="AmpR_promoter" ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 901 gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc ggtcgtacgt 961 ctccggattc gaattcggat ccgcggccgc atagataact gatccagtgt gctggaatta 1021 attcgctgtc tgcgagggcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga 1081 cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg 1141 cggtgatgcc tttgagggtg gccgcgtcca tctggtcaga aaagacaatc tttttgttgt 1201 caagcttgag gtgtggcagg cttgagatct ggccatacac ttgagtgaca atgacatcca 1261 ctttgccttt ctctccacag gtgtccactc ccaggtccaa ctgcaggtcg agcatgcatc 1321 tagggcggcc aattccgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc 1381 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt 1441 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt 1501 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg 1561 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca 1621 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg 1681 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc 1741 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct 1801 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta 1861 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata agcttgccac 1921 aacccgggat aattcctgca gccaatatgg gatcggccat tgaacaagat ggattgcacg 1981 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 2041 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 2101 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt 2161 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 2221 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 2281 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 2341 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 2401 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 2461 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga tgatctcgtc gtgacccatg 2521 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 2581 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 2641 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 2701 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga ggggatcaat 2761 tctctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa 2821 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta 2881 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 2941 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 3001 aacgcgtcga gtgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 3061 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt 3121 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 3181 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 3241 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 3301 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 3361 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 3421 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 3481 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 3541 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 3601 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 3661 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct 3721 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 3781 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 3841 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 3901 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 3961 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 4021 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 4081 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 4141 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 4201 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 4261 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 4321 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 4381 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 4441 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 4501 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 4561 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 4621 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 4681 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 4741 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 4801 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 4861 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 4921 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 4981 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 5041 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 5101 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 5161 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 5221 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc //
