pBudCE4.1-hrGFP哺乳双框质粒价格_品牌:钦诚生物-丁香通官网

保存条件：负20度

保质期：2年

供应商：钦诚生物

规格：5ug

基本信息

启动子:	CMV promoter,EF1-α promoter
复制子:	SV40 ori，ori
终止子:	bGH poly(A) signal
质粒分类:	哺乳系列质粒；哺乳表达质粒；哺乳双框质粒
质粒大小:	5303bp
质粒标签:	C-His,C-V5
原核抗性:	博来霉素Zeo（50μg/ml）
筛选标记:	博来霉素Zeo
克隆菌株:	DH5α等大肠杆菌
培养条件:	37℃，有氧，LB
表达宿主:	293T等哺乳细胞
培养条件:	37℃，5%CO2
诱导方式:	无需诱导，瞬时表达
5'测序引物:	CMV-F（CGCAAATGGGCGGTAGGCGTG） EF-1a- F (TCAAGCCTCAGACAGTGGTTC)
3'测序引物:	Bleo-R（CTGGATGATCCTCCAGCGC） BGH（TAGAAGGCACAGTCGAGG）

pBudCE4.1-hrGFP质粒是在pBudCE4.1载体中克隆进hrGFP绿色荧光基因。pBudCE4.1质粒使用时，E.coli 克隆菌株时最好使用不含Tn5基因的菌株，如TOP10和DH5αT1；采用电转法时最好用TOP10，博来霉素Zeocin 工作浓度推荐25 μg/ml ~50 μg/ml。
pBudCE4.1 载体设计用于从哺乳动物细胞中的单个载体独立表达两个基因。使用 pBudCE4.1 来产生稳定的哺乳动物表达细胞系可确保细胞中每个基因的拷贝数相等。这可以消除由于基因拷贝数的差异导致的表达变化。pBudCE4.1 可提供具有下列特征的表达盒：
• 用于高水平转录基因的 CMV，可选择用于进行快速检测的 c-myc 表位标签和用于进行简单纯化的 6xHis 序列
  • 用于高水平表达基因的 EF-1α 启动子，可选择用于快速检测的 V5 表位标签以及用于简单纯化的 6xHis 序列
  • 用于在哺乳动物细胞和大肠杆菌 (E. coli ) 中使用选择剂 Zeocin 进行高效选择的Sh ble (ZeoR) 基因
  pBudCE4.1 is a 4.6 kb vector designed for simultaneous expression of two genes in mammalian cell lines. The vector contains the human cytomegalovirus (CMV) immediate-early promoter and the human elongation factor 1α-subunit (EF-1α) promoter for high-level, constitutive, independent expression of two recombinant proteins (see page 13 for more information on the EF-1α promoter). Features of the vector allow detection and purification of expressed proteins (see pages 15–16) for more information). High-level stable and transient expression studies can be carried out in most mammalian cell types. In addition to the two promoters, the vector contains the following elements:
  • C-terminal peptides encoding the myc (c-myc) epitope or the V5 epitope and a polyhistidine (6xHis) metal-binding tag for detection and purification of recombinant proteins
  • Zeocin™ resistance gene for selection in E. coli and creation of stable, mammalian cell lines (Mulsant et al., 1988) (see pages 18–19 for more information)
  • SV40 origin for episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g., COS7) pBudCE4.1/lacZ/CAT is included for use as a positive control for transfection, expression, and detection in the cell line of choice.
  pBudCE4.1 is an improved version of pBudCE4. During construction of the original vector, an ATG was inadvertently created in the multiple cloning site (672–674 bp) 3′to the CMV promoter. Since it may interfere with proper translation of the cloned gene, this ATG was changed to ATT to create pBudCE4.1.

质粒图谱

质粒序列

LOCUS       Exported                5303 bp ds-DNA     circular SYN 21-七月-2016
DEFINITION synthetic circular DNA
KEYWORDS    pBudCE4.1-hrGFP
SOURCE      synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE   1 (bases 1 to 5303)
TITLE     Direct Submission
JOURNAL   Exported 2016年9月18日 from Plasmid
FEATURES             Location/Qualifiers
     source          1..5303
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        10..389
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        638..656
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             676..1395
                     /codon_start=1
                     /product="humanized Renilla green fluorescent protein"
                     /note="hrGFP"
                     /note="mammalian codon-optimized"
                     /translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
                     QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
                     DGGLVEIRSDINLIEEMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
                     QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
                     AQLTSLGKPLGSLHEWV"
     CDS             1427..1456
                     /codon_start=1
                     /product="Myc (human c-Myc oncogene) epitope tag"
                     /note="Myc"
                     /translation="EQKLISEEDL"
     CDS             1472..1489
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     polyA_signal    1520..1641
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     CDS             complement(1771..2145)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /note="BleoR"
                     /note="confers resistance to bleomycin, phleomycin, and
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(2164..2211)
                     /note="EM7 promoter"
                     /note="synthetic bacterial promoter "
     promoter        complement(2259..2575)
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      2273..2408
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     promoter        2597..3759
                     /note="EF-1-alpha promoter"
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     intron          2828..3750
                     /note="EF-1-alpha intron A"
                     /note="intron upstream of the start codon of human
                     EF-1-alpha"
     CDS             3835..3876
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /note="V5 tag"
                     /translation="GKPIPNPLLGLDST"
     CDS             3886..3903
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     polyA_signal    3932..4156
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(4288..4876)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
ORIGIN
        1 gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      481 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
      601 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
      661 cccaagcttg ccaccatggt gagcaagcag atcctgaaga acaccggcct gcaggagatc
      721 atgagcttca aggtgaacct ggagggcgtg gtgaacaacc acgtgttcac catggagggc
      781 tgcggcaagg gcaacatcct gttcggcaac cagctggtgc agatccgcgt gaccaagggc
      841 gcccccctgc ccttcgcctt cgacatcctg agccccgcct tccagtacgg caaccgcacc
      901 ttcaccaagt accccgagga catcagcgac ttcttcatcc agagcttccc cgccggcttc
      961 gtgtacgagc gcaccctgcg ctacgaggac ggcggcctgg tggagatccg cagcgacatc
     1021 aacctgatcg aggagatgtt cgtgtaccgc gtggagtaca agggccgcaa cttccccaac
     1081 gacggccccg tgatgaagaa gaccatcacc ggcctgcagc ccagcttcga ggtggtgtac
     1141 atgaacgacg gcgtgctggt gggccaggtg atcctggtgt accgcctgaa cagcggcaag
     1201 ttctacagct gccacatgcg caccctgatg aagagcaagg gcgtggtgaa ggacttcccc
     1261 gagtaccact tcatccagca ccgcctggag aagacctacg tggaggacgg cggcttcgtg
     1321 gagcagcacg agaccgccat cgcccagctg accagcctgg gcaagcccct gggcagcctg
     1381 cacgagtggg tgtaaatcga tcttaagcag tacttctaga ggatccgaac aaaaactcat
     1441 ctcagaagag gatctgaata tgcataccgg tcatcatcac catcaccatt gagtttgatc
     1501 cccgggaatt cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1561 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1621 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1681 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1741 ggcggtggaa tcgaaatctc gtagcacgtg tcagtcctgc tcctcggcca cgaagtgcac
     1801 gcagttgccg gccgggtcgc gcagggcgaa ctcccgcccc cacggctgct cgccgatctc
     1861 ggtcatggcc ggcccggagg cgtcccggaa gttcgtggac acgacctccg accactcggc
     1921 gtacagctcg tccaggccgc gcacccacac ccaggccagg gtgttgtccg gcaccacctg
     1981 gtcctggacc gcgctgatga acagggtcac gtcgtcccgg accacaccgg cgaagtcgtc
     2041 ctccacgaag tcccgggaga acccgagccg gtcggtccag aactcgaccg ctccggcgac
     2101 gtcgcgcgcg gtgagcaccg gaacggcact ggtcaacttg gccatggttt agttcctcac
     2161 cttgtcgtat tatactatgc cgatatacta tgccgatgat taattgtcaa cacgtgctga
     2221 tcagatccga aaatggatat acaagctccc gggagctttt tgcaaaagcc taggcctcca
     2281 aaaaagcctc ctcactactt ctggaatagc tcagaggcag aggcggcctc ggcctctgca
     2341 taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg
     2401 ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt
     2461 tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg
     2521 agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctcgt
     2581 cgagctagct tcgtgaggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
     2641 ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg
     2701 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
     2761 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
     2821 aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc
     2881 ttgcgtgcct tgaattactt ccacctggct ccagtacgtg attcttgatc ccgagctgga
     2941 gccaggggcg ggccttgcgc tttaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
     3001 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
     3061 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
     3121 caagatagtc ttgtaaatgc gggccaggat ctgcacactg gtatttcggt ttttgggccc
     3181 gcggccggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
     3241 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
     3301 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     3361 gttgcgtgag cggaaagatg gccgcttccc ggccctgctc cagggggctc aaaatggagg
     3421 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     3481 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     3541 tagttctgga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     3601 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     3661 ttctcgttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     3721 gtggttcaaa gtttttttct tccatttcag gtgtcgtgaa cacgtggtcg cggccgcttc
     3781 gaaggtacca gcacagtgga ctcgagagat ctggccggct gggcccgttt cgaaggtaag
     3841 cctatcccta accctctcct cggtctcgat tctacgcgta ccggtcatca tcaccatcac
     3901 cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     3961 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     4021 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     4081 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     4141 gcggtgggct ctatggcttc tgaggcggaa agaaccagtg gcggtaatac ggttatccac
     4201 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4261 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4321 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4381 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4441 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4501 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4561 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4621 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4681 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     4741 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4801 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4861 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4921 cgaaaactca cgttaaggga ttttggtcat gacattaacc tataaaaata ggcgtatcac
     4981 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     5041 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     5101 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     5161 tgtactgaga gtgcaccata tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     5221 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     5281 cgggcctctt cgctattacg cca