pCDNA3.0-HA-N哺乳表达质粒

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
复制子:	pUC ori，F1 ori
终止子:	bGH poly(A) signal
质粒分类:	哺乳细胞真核表达载体；蛋白过表达质粒
质粒大小:	5484bp
质粒标签:	N-HA
原核抗性:	氨苄青霉素(Ampicillin)
筛选标记:	新霉素(Neomycin)/遗传霉素(Geneticin/G418)
克隆菌株:	DH5α
培养条件:	37℃，有氧，LB
表达宿主:	哺乳细胞
诱导方式:	无需诱导
5'测序引物:	T7：TAATACGACTCACTATAGGG
3'测序引物:	SP6：ATTTAGGTGACACTATAGAA
备注:	载体上有起始密码子ATG，注意不要移码。

质粒简介

        pCDNA3.0-HA-N载体是专门设计用来在哺乳细胞中进行高水平的稳定表达和瞬时表达。其中人巨细胞病毒早期启动子(CMV)在广泛的哺乳动物细胞中可以实现高表达。新霉素抗性基因可以用来筛选稳定细胞系，具体用G418筛选，另外HA做为标签能在WB实验中更方便的检测到目的基因是否表达。
        pcDNA3.0-HA-N is 5.4 kb vectors  designed for high-level stable and transient expression in mammalian hosts. High-level stable and non-replicative transient expression can be carried out in most mammalian cells. The vectors contain the following elements:
        Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells.
        Neomycin resistance gene for selection of stable cell lines.
        HA epitope for Western blot experiment.

质粒图谱

质粒序列

LOCUS       Exported                5484 bp ds-DNA     circular SYN 20-NOV-2016
DEFINITION  synthetic circular DNA
FEATURES             Location/Qualifiers
     source          1..5484
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        235..614
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        863..881
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             898..924
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /note="HA"
                     /translation="YPYDVPDYA"
     misc_feature    927..1032
                     /note="MCS"
     promoter        complement(1036..1054)
                     /note="SP6 promoter"
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     polyA_signal    1080..1304
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1350..1778
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        1792..2122
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      1973..2108
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             2189..2983
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3157..3278
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3327..3343)
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3351..3367
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(3375..3405)
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    3420..3441
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3729..4317)
                     /direction=LEFT
                     /note="pUC ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4488..5348)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5349..5453)
                     /gene="bla"
                     /note="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 ggagacccgc caccatgtac
      901 ccatacgacg tcccagacta cgctggaagc ttggtaccga gctcggatcc actagtaacg
      961 gccgccagtg tgctggaatt ctgcagatat ccatcacact ggcggccgct cgagcatgca
     1021 tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac
     1081 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1141 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1201 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1261 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     1321 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     1381 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1441 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1501 tcggggcatc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1561 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1621 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1681 ccctatctcg gtctattctt ttgatttata agggattttg gggatttcgg cctattggtt
     1741 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     1801 ttagggtgtg gaaagtcccc aggctcccca ggcaggcaga agtatgcaaa gcatgcatct
     1861 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     1921 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     1981 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     2041 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     2101 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     2161 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     2221 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     2281 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     2341 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     2401 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     2461 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     2521 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     2581 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     2641 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     2701 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     2761 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     2821 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     2881 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     2941 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     3001 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     3061 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     3121 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     3181 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     3241 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     3301 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3361 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3421 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3481 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3541 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3601 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3661 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3721 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3781 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3841 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3901 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     3961 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4021 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4081 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4141 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4201 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4261 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4321 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4381 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4441 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4501 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4561 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4621 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4681 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4741 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4801 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4861 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4921 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4981 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5041 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5101 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5161 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5221 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5281 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5341 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5401 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5461 ccccgaaaag tgccacctga cgtc