pHB201枯草芽孢杆菌表达载体

基本信息
启动子: T7,T3
复制子: ori
终止子: T1
质粒分类: 广宿主系列，枯草杆菌载体
质粒大小: 6594bp
原核抗性: Chl
克隆菌株: DH5α
培养条件: 37℃，有氧 LB
表达宿主: 枯草芽孢杆菌
诱导方式: IPTG诱导
5'测序引物: T7:TAATACGACTCACTATAGGG
3'测序引物: 根据序列设计引物
质粒简介
    A genomic library of Bacillus lyticus was constructed in lambda GEM 11 vector and screened for the xylanase gene using Congo red plate assay. A 16-kb fragment containing the xylanase gene was obtained which was further subcloned using Mbo I partial digestion in an E. coli pUC 19 vector. A 1.3-kb sub-fragment was obtained which coded for a xylanase gene of Mr 23,650 Da. This fragment was sequenced and the homology was checked with known xylanases. The maximum homology was 97%, which was obtained with an endo xylanase gene from Bacillus species at the DNA level, while the translated sequence showed only one amino acid change from alanine to serine at position number 102. Expression was checked in E. coli, using the native promoter, and an extracellular activity of 5.25 U/mL was obtained. Cloning of the gene was done in Bacillus subtilis using a shuttle vector pHB 201, which resulted in increasing the basal level xylanase activity from 14.02 to 22.01 U/mL.

质粒图谱
质粒序列
LOCUS       Exported File           6594 bp ds-DNA    circular SYN 29-5-2015
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    pHB201
SOURCE      synthetic DNA construct
 ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6594)
 AUTHORS   .
 TITLE     Direct Submission
 JOURNAL   Exported 2015-5-29 from  http://www.biovector.net
FEATURES             Location/Qualifiers
    source          1..6594
                    /organism="synthetic DNA construct"
                    /mol_type="other DNA"
    promoter        1533..1551
                    /note="T3 promoter"
                    /note="promoter for bacteriophage T3 RNA polymerase"
    misc_feature    1564..1671
                    /note="MCS"
                    /note="pBluescript multiple cloning site"
    primer_bind     1588..1604
                    /note="SK primer"
                    /note="common sequencing primer, one of multiple similar
                    variants"
    primer_bind     complement(1638..1654)
                    /note="KS primer"
                    /note="common sequencing primer, one of multiple similar
                    variants"
    promoter        complement(1680..1698)
                    /note="T7 promoter"
                    /note="promoter for bacteriophage T7 RNA polymerase"
    primer_bind     complement(1711..1727)
                    /note="M13 fwd"
                    /note="common sequencing primer, one of multiple similar
                    variants"
    terminator      2073..2159
                    /gene="Escherichia coli rrnB"
                    /note="rrnB T1 terminator"
                    /note="transcription terminator T1 from the E. coli rrnB
                    gene"
    terminator      2251..2278
                    /note="rrnB T2 terminator"
                    /note="transcription terminator T2 from the E. coli rrnB
                    gene"
    CDS             complement(2717..3367)
                    /codon_start=1
                    /note="CamR"
                    /translation="MNFNKIDLDNWKRKEIFNHYLNQQTTFSITTEIDISVLYRNIKQE
                    GYKFYPAFIFLVTRVINSNTAFRTGYNSDGELGYWDKLEPLYTIFDGVSKTFSGIWTPV
                    KNDFKEFYDLYLSDVEKYNGSGKLFPKTPIPENAFSLSIIPWTSFTGFNLNINNNSNYL
                    LPIITAGKFINKGNSIYLPLSLQVHHSVCDGYHAGLFMNSIQELSDRPNDWLL"
    rep_origin      6329..323
                    /direction=RIGHT
                    /note="ori"
                    /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                    replication"
ORIGIN
       1 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
      61 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     121 gtgagcattg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     181 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     241 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     301 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     361 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     421 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     481 agtcagtgag cgaggaagcg gaagaagctt actttattgt tttgccattt taaaaggttc
     541 aaataaaata tttttcataa aattatcaga tgttttttaa aaaatgaaat aaaagaagaa
     601 aaaaatagaa aatacactaa cccaaagaag cgcgtaatat cgagggttta agagacaata
     661 agaaaaaaag attgaaaaat gacattaaat tcttgacagg gagagatagg tttgatagaa
     721 tataatagtt gtcgcgagag acgacgttga agcgcgacaa gctagaccat tgaaaactga
     781 ataaagaaga atgactcatg tgatgtagca atacatcaaa aatctgtcaa tcaataatga
     841 aagacaagcc atcaattcgg tgacttaaat actttatttg agagtttgat cctctagagt
     901 cgacctgcag gctttaacgt aggcaaagct cagggtagac tttgaatgga cagaaacatg
     961 acatatctct tgaaaggatg attgtggtgg tgaaaacaga taaaatctcc tcctgaatac
    1021 agtaaatcac attcaggagg agataaaatt gtttaaacaa atagacgaaa attatctgcg
    1081 aaaagagcac tttcaccatt atatgacgtt aacccgatgc tcatatagct tggtgatcaa
    1141 tctagacatc acgaaattgc atgcaatatt aaaagaaaaa aagctgaaag tatatcctgt
    1201 gcaaatttat ttgttagcaa gagctgtgca aaaaattcct gagtttcgga tggatcaagt
    1261 gaacgatgaa cttggttact gggagattct ccatcctagt tatacgattc taaataaaga
    1321 aacaaagacg ttttcaagta tttggacgcc ttttgatgaa aactttgctc agttttataa
    1381 aagctgtgta gccgatattg aaacatttag caaaagcagc aacctatttc cgaaacctca
    1441 tatgccagaa aacatgttca atatttcaag tctaccgtgg attgatttta cttcttttaa
    1501 ccttaatgta tctacagatg aagctagcgc gcaattaacc ctcactaaag ggaacaaaag
    1561 ctggagctcc accgcggtgg cggccgctct agaactagtg gatcccccgg gctgcaggaa
    1621 ttcgatatca agcttatcga taccgtcgac ctcgaggggg ggcccggtac ccaattcgcc
    1681 ctatagtgag tcgtattacg cgcgcaattc actggccgtc gttttacaac gtcgtgactg
    1741 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
    1801 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatta
    1861 gagatcaatt ccctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc
    1921 agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc
    1981 acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc
    2041 tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag
    2101 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
    2161 cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc
    2221 cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg
    2281 cgtttctaca aactcttcct gtcgtcatat ctacaagcca tccccccaca gatacggtaa
    2341 actagcctcg tttttgcatc aggaaagcag ggaattgatc tgatgggtag aaccttgggc
    2401 tttgcccaaa cccaccaacg gatggccgtt ggattccaac attcaatccc cccaaccccc
    2461 cgcatctccg gggggctccc tcgccggttg gcgtcaaaca aacgagtttg tttgagtgct
    2521 gaaagggttc gggtagcact gagcgagtca actccttaat cctccctacg gtccggttac
    2581 cgttgactca cgccataccc atcgaatggc gaatggcgga tctggagctg taatataaaa
    2641 accttcttca actaacgggg caggttagtg acattagaaa accgactgta aaaagtacag
    2701 tcggcattat ctcatattat aaaagccagt cattaggcct atctgacaat tcctgaatag
    2761 agttcataaa caatcctgca tgataaccat cacaaacaga atgatgtacc tgtaaagata
    2821 gcggtaaata tattgaatta cctttattaa tgaattttcc tgctgtaata atgggtagaa
    2881 ggtaattact attattattg atatttaagt taaacccagt aaatgaagtc catggaataa
    2941 tagaaagaga aaaagcattt tcaggtatag gtgttttggg aaacaatttc cccgaaccat
    3001 tatatttctc tacatcagaa aggtataaat cataaaactc tttgaagtca ttctttacag
    3061 gagtccaaat accagagaat gttttagata caccatcaaa aattgtataa agtggctcta
    3121 acttatccca ataacctaac tctccgtcgc tattgtaacc agttctaaaa gctgtatttg
    3181 agtttatcac ccttgtcact aagaaaataa atgcagggta aaatttatat ccttcttgtt
    3241 ttatgtttcg gtataaaaca ctaatatcaa tttctgtggt tatactaaaa gtcgtttgtt
    3301 ggttcaaata atgattaaat atctcttttc tcttccaatt gtctaaatca attttattaa
    3361 agttcatttg atatgcctcc taaattttta tctaaagtga atttaggagg cttacttgtc
    3421 tgctttcttc attagaatca atcctttttt aaaagtcaat attactgtaa cataaatata
    3481 tattttaaaa atatcccact ttatccaatt ttcgtttgtt gaactaatgg gtgctttagt
    3541 tgaagaataa agaccacatt aaaaaatgtg gtcttttgtg tttttttaaa ggatttgagc
    3601 gtagcgaaaa atccttttct ttcttatctt gataataagg gtaactattg ccgcgcctga
    3661 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataagc cggtcttttt
    3721 cgattttggc gaagccgaaa tcgggtcttt tcttatcttg atactatata gaaacaacat
    3781 catttttcaa aattaggtca aagccttgtg tatcaagggt ttgatggttc tttgacaggt
    3841 aaaaactcct tctgctatta ttaaggtgtc gaatcaaaat aatagaatgc tagagaacta
    3901 gctcagaagg agtttttttg ttgatttatt catctgaaaa tgattatagc atcctcgaag
    3961 ataaaaccgc aacaggtaaa aagcgggatt ggaaggggaa aaagagacgg acgaacctca
    4021 tggcggagca ttacgaagcg ttagagagta agattggggc accttactat ggcaaaaagg
    4081 ctgaaaaact aattagttgt gcagagtatc tttcgtttaa gagagacccg gagacgggca
    4141 agttaaaact gtatcaagcc catttttgta aagtgaggtt atgtccgatg tgtgcgtggc
    4201 gcaggtcgtt aaaaattgct tatcacaata agttgatcgt agaggaagcc aatagacagt
    4261 acggctgcgg atggattttt ctcacgctga cgattcgaaa tgtaaaggga gaacggctga
    4321 agccacaaat ttctgcgatg atggaaggct ttaggaaact gttccagtac aaaaaagtaa
    4381 aaacttcggt tcttggattt ttcagagctt tagagattac caaaaatcat gaagaagata
    4441 catatcatcc tcattttcat gtgttgatac cagtaaggaa aaattatttt gggaaaaact
    4501 atattaagca ggcggagtgg acgagccttt ggaaaaaggc gatgaaattg gattacactc
    4561 caattgtcga tattcgtcga gtgaaaggta aagctaagat tgacgctgaa cagattgaaa
    4621 acgatgtgcg gaacgcaatg atggagcaaa aagctgttct cgaaatctct aaatatccgg
    4681 ttaaggatac ggatgttgtg cgcggtaata aggtgactga agacaatctg aacacggtgc
    4741 tttacttgga tgatgcgttg gcagctcgaa ggttaattgg atacggtggc attttgaagg
    4801 agatacataa agagctgaat cttggtgatg cggaggacgg cgatctggtc aagattgagg
    4861 aagaagatga cgaggttgca aatggtgcat ttgaggttat ggcttattgg catcctggca
    4921 ttaaaaatta cataatcaaa taaaaaaagc agacctttag aaggcctgct tttttaacta
    4981 acccatttgt attgtgttga aatatgtttt gtatggtgca ctctcagtac aatctgctct
    5041 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
    5101 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
    5161 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
    5221 ctatttttat aggttaatgt catgataata atggtttctt agcgattcac aaaaaatagg
    5281 cacacgaaaa acaagttaag ggatgcagtt tatgcatccc ttaacttact tattaaataa
    5341 tttatagcta ttgaaaagag ataagaattg ttcaaagcta atattgttta aatcgtcaat
    5401 tcctgcatgt tttaaggaat tgttaaattg attttttgta aatattttct tgtattcttt
    5461 gttaacccat ttcataacga aataattata cttttgttta tctttgtgtg atattcttga
    5521 tttttttcta cttaatctga taagtgagct attcacttta ggtttaggat gaaaatattc
    5581 tcttggaacc atacttaata tagaaatatc aacttctgcc attaaaagta atgccaatga
    5641 gcgttttgta tttaataatc ttttagcaaa cccgtattcc acgattaaat aaatctcatt
    5701 agctatacta tcaaaaacaa ttttgcgtat tatatccgta cttatgttat aaggtatatt
    5761 accatatatt ttataggatt ggtttttagg aaatttaaac tgcaatatat ccttgtttaa
    5821 aacttggaaa ttatcgtgat caacaagttt attttctgta gttttgcata atttatggtc
    5881 tatttcaatg gcagttacga aattacacct ctttactaat tcaagggtaa aatggccttt
    5941 tcctgagccg atttcaaaga tattatcatg ttcatttaat cttatatttg tcattatttt
    6001 atctatatta tgttttgaag taataaagtt ttgactgtgt tttatatttt tctcgttcat
    6061 tataaccctc tttaatttgg ttatatgaat tttgcttatt aacgattcat tataaccact
    6121 tattttttgt ttggttgata atgaactgtg ctgattacaa aaatactaaa aatgcccata
    6181 ttttttcctc cttataaaat tagtataatt atagcacgaa aaggatctag gtgaagatcc
    6241 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
    6301 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
    6361 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
    6421 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
    6481 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
    6541 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt ctta
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