pIRESneo2

pIRESneo2

pIRES-Neo2载体基本信息

出品公司:	Ybscience
载体名称:	pIRESneo2, pIRES-neo2, pIRES neo2
质粒类型:	哺乳动物细胞表达载体
启动子:	CMV IE
表达水平:	高
克隆方法:	多克隆位点，限制性内切酶
载体大小:	5295bp
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
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:
稳定性:	稳表达 或瞬表达
组成型:	组成型
病毒/非病毒:	非病毒

 

pIRES-Neo2载体质粒图谱和多克隆位点信息

pIRES-Neo2载体简介

Description:
pIRESneo2 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 moving the initiation codon of the neomycin phosphotransferase gene to a less optimal position for translation as directed by the IRES sequence (attenuated IRES; 1). The expression cassette of pIRESneo2 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 of the bovine growth hormone. 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 pIRESneo2 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 mg/ml G418 (#8056-1) 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.

pIRES-Neo2载体序列

LOCUS       pIRESneo2	5295 bp 	DNA     circular	SYN
DEFINITION  pIRESneo2
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
COMMENT     This file is created by Vector NTI
            http://www.biofeng.com/
COMMENT     VNTAUTHORNAME|biofeng.com|
FEATURES             Location/Qualifiers
     source          1..5295
                     /organism="pIRESneo2"
                     /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"
     misc_feature    complement(2782..2799)
                     /label="BGH_rev_primer"
     terminator      2785..3012
                     /label="bGH_PA_terminator"
     misc_feature    3059..3078
                     /label="EBV_rev_primer"
     promoter        complement(3134..3152)
                     /label="M13_reverse_primer"
     misc_feature    complement(3151..3173)
                     /label="M13_pUC_rev_primer"
     promoter        complement(3187..3216)
                     /label="lac_promoter"
     rep_origin      complement(3525..4144)
                     /label="pBR322_origin"
     gene            complement(4299..5159)
                     /label="Ampicillin"
                     /gene="Ampicillin"
     CDS             complement(4299..5159)
                     /label="ORF frame 2"
     promoter        complement(5201..5229)
                     /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 ATGTGATTTT 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 TCTCTAGAGC TCGCTGATCA GCCTCGACTG TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT
 2821 GCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC TCCCACTGTC CTTTCCTAAT
 2881 AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCA TTCTATTCTG GGGGGTGGGG
 2941 TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG CAGGCATGCT GGGGATGCGG
 3001 TGGGCTCTAT GGCTTCTGAG GCGGAAAGAA CCAGCTGGGG CTCGAGTGCA TTCTAGTTGT
 3061 GGTTTGTCCA AACTCATCAA TGTATCTTAT CATGTCTGTA TACCGTCGAC CTCTAGCTAG
 3121 AGCTTGGCGT AATCATGGTC ATAGCTGTTT CCTGTGTGAA ATTGTTATCC GCTCACAATT
 3181 CCACACAACA TACGAGCCGG AAGCATAAAG TGTAAAGCCT GGGGTGCCTA ATGAGTGAGC
 3241 TAACTCACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC
 3301 CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG GTTTGCGTAT TGGGCGCTCT
 3361 TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA
 3421 GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC
 3481 ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT
 3541 TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG
 3601 CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC
 3661 TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC
 3721 GTGGCGCTTT CTCAATGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC
 3781 AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC
 3841 TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT
 3901 AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT
 3961 AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC
 4021 TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT
 4081 TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG
 4141 ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC
 4201 ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA
 4261 TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG
 4321 GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG
 4381 TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA
 4441 GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG
 4501 CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA
 4561 GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC
 4621 ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA
 4681 AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG
 4741 ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT
 4801 AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC
 4861 AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG
 4921 GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG
 4981 GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT
 5041 GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA
 5101 GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA
 5161 CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC
 5221 ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA
 5281 GTGCCACCTG ACGTC
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