| 出品公司: | Clontech |
|---|---|
| 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: | / |
| 载体抗性: | 氨苄 |
| 筛选标记: | Neomycin/ G418 |
| 备注: | 需要将你的基因克隆到 IRES-neo 元件的上游。 |
| 产品目录号: | 6938-1 |
| 稳定性: | 稳表达 Stable |
| 组成型: | 组成型 Constitutive |
| 病毒/非病毒: | 非病毒 |
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pIRES-Neo2载体基本信息
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.biovector.net/
COMMENT VNTAUTHORNAME|biovector.net|
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
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