pIRESneo3

价格:800元

联系方式:I47-825O-882O

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pIRESneo 3载体基本信息

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:
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:
载体抗性: 氨苄青霉素(Ampicillin)
筛选标记: Neomycin_1.html' target='_blank'>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

pIRESneo 3载体质粒图谱和多克隆位点信息

pIRESneo3载体图谱和多克隆位点

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
//

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