pBR322大肠克隆质粒

基本信息

复制子:	PBR322 ori
质粒分类:	大肠系列质粒；大肠克隆质粒；基因克隆质粒
质粒大小:	4361bp
原核抗性:	氨苄青霉素Amp（100μg/ml），四环素Tet（10μg/ml）
克隆菌株:	DH5α等大肠杆菌
培养条件:	37℃，有氧，LB
5'测序引物:	PBR322.F（AAGTGCCACCTGACGTCTAA）
3'测序引物:	PBR322.R (GCCTGCCACCATACCCAC )
备注:	应用很广的基因克隆质粒

质粒简介

        pBR322质粒相对分子质量较小，它带有一个复制起始位点，保证了该质粒只在大肠杆菌的细胞中行使复制的功能。具有两种抗生素抗性基因——氨苄青霉素抗性基因和四环素抗性基因，可供转化子的选择标记。具有较高的拷贝数，经过氯霉素扩增以后，每个细胞中可累积1000-3000份拷贝，该特性为重组体DNA的制备提供了极大的方便。同时，由于其为人工构建质粒，虽然带有抗药性基因，但已不能在自然界的宿主细胞间转移，同时应用安全菌株，亦不会引起抗生素抗性基因传播。
        pBR322是一个人工构建的重要质粒，有万能质粒之称。它是由pSF2124、pMB1及pSC101三个亲本质粒经复杂的重组过程构建而成的。质粒载体pBR322是研究得最多，使用最早且应用最广泛的大肠杆菌质粒载体之一。符号质粒载体pBR322中的“p代表质粒；“BR”代表两位两位研究者Bolivar和Rogigerus姓氏的字首，“322”是实验编号。
        质粒载体pBR322的大小为4361bp，相对分子质量较小的是它第一个优点。优点之二是它带有一个复制起始位点，保证了该质粒只在大肠杆菌的细胞中行使复制的功能。具有两种抗生素抗性基因——氨苄青霉素抗性基因和四环素抗性基因，可供转化子的选择标记是它的第三个优点。

质粒图谱

质粒序列

LOCUS       Exported                4361 bp ds-DNA    circular SYN 22-9-2015
KEYWORDS    Untitled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4361)
  AUTHORS   admin
  TITLE     Direct Submission
  JOURNAL   Exported 2015-9-22  
FEATURES             Location/Qualifiers
     source          1..4361
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        10..38
                     /note="tet promoter"
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     CDS             86..1276
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /note="TcR"
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     CDS             1915..2106
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    2208..2348
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(2534..3122)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(3293..4153)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4154..4258)
                     /gene="bla"
                     /note="AmpR promoter"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata
      241 tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg
      301 ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac
      361 cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac
      421 aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca
      481 cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg
      541 actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct
      601 caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaccgat
      661 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
      721 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
      781 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
      841 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
      901 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt
      961 cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc
     1021 cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca
     1081 tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc
     1141 gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat
     1201 tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg
     1261 ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga
     1321 attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac
     1381 atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg
     1441 tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg
     1501 ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc
     1561 tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg
     1621 taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca
     1681 ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga
     1741 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa
     1801 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt
     1861 ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc
     1921 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt
     1981 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac
     2041 gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     2101 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     2161 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     2221 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
     2281 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     2341 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2401 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2461 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2521 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2581 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2641 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2701 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2761 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2821 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2881 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2941 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     3001 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3061 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3121 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3181 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3241 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3301 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3361 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3421 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3481 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3541 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3601 ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3661 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3721 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3781 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3841 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3901 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3961 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4021 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4081 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4141 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4201 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4261 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     4321 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga a