pHIS1525

价格:1500元

联系方式:I47-825O-882O

相关技术服务:质粒构建    基因合成    质粒大提

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载体基本信息

出品公司: --
载体名称: pHIS1525
质粒类型: 巨大芽孢杆菌表达载体
高拷贝/低拷贝: --
启动子: --
克隆方法: 多克隆位点,限制性内切酶
载体大小: 7402 bp
5' 测序引物及序列: --
3' 测序引物及序列: --
载体标签: 6xHis
载体抗性: 氨苄青霉素四环素
筛选标记: 四环素
备注: --
产品目录号: --
稳定性: --
组成型: --
病毒/非病毒: --

载体图谱质粒图谱和多克隆位点信息

pHIS1525载体图谱



载体简介

Plasmids pHIS1522/pHIS1525 both contain the strong xylA promoter (PxylA)
originating from Bacillus megaterium. Transcription from this promoter is xylose
inducible (after xylose addition, the xylose repressor coded by the xylR gene on
the plasmids is released from PxylA and transcription is initiated). The most
convenient cloning sites for insertion of DNA fragments carrying heterologous
genes are located in an open reading frame (orf1) under control of the xyloseinducible
promoter PxylA (15 unique restriction sites; see sequence). Therefore, any
protein can be expressed using one out of three functionally different fusion
strategies.
A transcriptional fusion requires that the gene of interest carries its own ribosome
binding sequence (RBS) and translation initiation codon. Such a DNA fragment
can be fused into any of the available restriction sites within orf1. Whether the
resulting transcriptional fusion leads to expression of the gene of interest, which
is independent from orf1 expression, depends on the location of the created orf1
stop codon with respect to the start codon of the gene of interest. lf these are close
together, translational coupling may occur, in which the ribosomes translating the
orf1 reading frame would terminate at its stop codon, creating a locally high
concentration of ribosomes, so that translation initiation at the new start codon
would be more efficient compared to a construct in which the orf1 translation
terminates farther away from the start codon.
On the other hand, if the orf1 reading frame continues for a long distance into the
reading frame of the gene of interest, the ribosomes translating the created orf1
fusion protein might inhibit initiation of translation of the protein of interest.
Therefore, it is advisable to pay attention to placement of a stop codon when
constructing the gene fusion. Taken together, although a transcriptional or operon
fusion is constructed, the efficient translation of the orf1 reading frame, and any
fusion thereof created by the insertion, is likely to, positively or negatively,
influence the translation efficiency of the gene of interest.
Alternatively, a truncated version of the gene of interest, lacking its own start
codon, may be fused in frame to the orf1 reading frame on pHIS1522/pHIS1525
to create a translational or protein fusion. This will result in expression of a chimeric
protein consisting of up to 18 amino acids specified by the orf1 encoding
sequence, followed by the sequence encoded by the gene of interest.
Using the BsrGI restriction site directly before the ATG start codon enables cloning
without changing the N-terminus of the protein of interest. pHIS1525 contains a
sequence encoding the B. subtilis extracellular esterase (LipA) and allows cloning
of target genes directly after the signal peptidase restriction site using the Kas I,
Nar I, or SfoI restriction sites.
It is important to note that the multiple cloning site and its reading frame are
identical in pHIS1522 and pHIS1525 starting from BglII. Hence, a parallel
cloning strategy of the gene of interest in pHIS1522 for intracellular and in
pHIS1525 for extracellular production is possible. The 6xHis sequence upstream
the MCS allows convenient purification and detection of the expressed Histagged
target proteins. For expression of target proteins without a 6xHis tag,
suitable plasmid constructs (pMM1522 & pMM1525) are also available.

载体序列

LOCUS       pHis1525                7474 bp DNA     circular SYN 03-NOV-2022
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7474)
  AUTHORS   Ming
  TITLE     Direct Submission
  JOURNAL   Exported Nov 3, 2022 from SnapGene 6.0.2 to Vector NTI(R) format
            https://www.biofeng.com
COMMENT     VNTDATE|1001116800|
COMMENT     VNTDBDATE|1001116800|
COMMENT     VNTNAME|pHis1525|
COMMENT     VNTAUTHORNAME|Ming|
FEATURES             Location/Qualifiers
     CDS             18..35
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
     rep_origin      complement(176..764)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(935..1795)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
     promoter        complement(1796..1900)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(2064..3440)
                     /codon_start=1
                     /gene="Bacillus subtilis tetL"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MNTSYSQSNLRHNQILIWLCILSFFSVLNEMVLNVSLPDIANDFN
                     KPPASTNWVNTAFMLTFSIGTAVYGKLSDQLGIKRLLLFGIIINCFGSVIGFVGHSFFS
                     LLIMARFIQGAGAAAFPALVMVVVARYIPKENRGKAFGLIGSIVAMGEGVGPAIGGMIA
                     HYIHWSYLLLIPMITIITVPFLMKLLKKEVRIKGHFDIKGIILMSVGIVFFMLFTTSYS
                     ISFLIVSVLSFLIFVKHIRKVTDPFVDPGLGKNIPFMIGVLCGGIIFGTVAGFVSMVPY
                     MMKDVHQLSTAEIGSVIIFPGTMSVIIFGYIGGILVDRRGPLYVLNIGVTFLSVSFLTA
                     SFLLETTSWFMTIIIVFVLGGLSFTKTVISTIVSSSLKQQEAGAGMSLLNFTSFLSEGT
                     GIAIVGGLLSIPLLDQRLLPMEVDQSTYLYSNLLLLFSGIIVISWLVTLNVYKHSQRDF
                     "
     CDS             complement(3640..4644)
                     /codon_start=1
                     /gene="repB"
                     /product="RepB replication protein"
                     /label=repB
                     /note="from Enterococcus faecalis plasmid pAM-alpha-1"
                     /db_xref="GI:22652809"
                     /protein_id="AAN03827.1"
     promoter        5069..5097
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ccggcctgag aggatcgcat caccatcacc atcactaacc ggtccaagaa ttggagccaa
       61 tcaattcttg cggagaactg tgaatgcgca aaccaaccct tggcagaaca tatccatcgc
      121 gtccgccatc tccagcagcc gcacgcggcg catctcgggc cgcgttgctg gcgtttttcc
      181 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
      241 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
      301 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
      361 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
      421 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
      481 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
      541 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
      601 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
      661 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
      721 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
      781 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
      841 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
      901 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
      961 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     1021 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     1081 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     1141 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     1201 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg
     1261 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     1321 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     1381 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     1441 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     1501 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata
     1561 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     1621 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     1681 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     1741 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     1801 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     1861 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     1921 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     1981 cgaggccctt tcgtcttcaa gaattcctgt tataaaaaaa ggatcaattt tgaactctct
     2041 cccaaagttg atcccttaac gatttagaaa tccctttgag aatgtttata tacattcaag
     2101 gtaaccagcc aactaatgac aatgattcct gaaaaaagta ataacaaatt actatacaga
     2161 taagttgact gatcaacttc cataggtaac aacctttgat caagtaaggg tatggataat
     2221 aaaccaccta caattgcaat acctgttccc tctgataaaa agctggtaaa gttaagcaaa
     2281 ctcattccag caccagcttc ctgctgtttc aagctacttg aaacaattgt tgatataact
     2341 gttttggtga acgaaagccc acctaaaaca aatacgatta taattgtcat gaaccatgat
     2401 gttgtttcta aaagaaagga agcagttaaa aagctaacag aaagaaatgt aactccgatg
     2461 tttaacacgt ataaaggacc tcttctatca acaagtatcc caccaatgta gccgaaaata
     2521 atgacactca ttgttccagg gaaaataatt acacttccga tttcggcagt acttagctgg
     2581 tgaacatctt tcatcatata aggaaccata gagacaaacc ctgctactgt tccaaatata
     2641 attcccccac aaagaactcc aatcataaaa ggtatatttt tccctaatcc gggatcaaca
     2701 aaaggatctg ttactttcct gatatgtttt acaaatatca ggaatgacag cacgctaacg
     2761 ataagaaaag aaatgctata tgatgttgta aacaacataa aaaatacaat gcctacagac
     2821 attagtataa ttcctttgat atcaaaatga ccttttatcc ttacttcttt ctttaataat
     2881 ttcataagaa acggaacagt gataattgtt atcataggaa tgagtagaag ataggaccaa
     2941 tgaatataat gggctatcat tccaccaatc gctggaccga ctccttctcc catggctact
     3001 atcgatccaa taagaccaaa tgctttaccc ctattttcct ttggaatata gcgcgcaact
     3061 acaaccatta cgagtgctgg aaatgcagct gcaccagccc cttgaataaa acgagccata
     3121 ataagtaagg aaaagaaaga atggccaaca aacccaatta ccgacccgaa acaatttatt
     3181 ataattccaa ataggagtaa ccttttgatg cctaattgat cagatagctt tccatataca
     3241 gctgttccaa tggaaaaggt taacataaag gctgtgttca cccagtttgt actcgcaggt
     3301 ggtttattaa aatcatttgc aatatcaggt aatgagacgt tcaaaaccat ttcatttaat
     3361 acgctaaaaa aagataaaat gcaaagccaa attaaaattt ggttgtgtcg taaattcgat
     3421 tgtgaatagg atgtattcac atttcaccct ccaataatga gggcagacgt agtttatagg
     3481 gttaatgata cgcttccctc ttttaattga accctgttac attcattaca cttcataatt
     3541 aattcctcct aaacttgatt aaaacatttt accacatata aactaagttt taaattcagt
     3601 atttcatcac ttatacaaca atatggcccg tttgttgaac tactctttaa taaaataatt
     3661 tttccgttcc caattccaca ttgcaataat agaaaatcca tcttcatcgg ctttttcgtc
     3721 atcatctgta tgaatcaaat cgccttcttc tgtgtcatca aggtttaatt ttttatgtat
     3781 ttcttttaac aaaccaccat aggagattaa ccttttacgg tgtaaacctt cctccaaatc
     3841 agacaaacgt ttcaaattct tttcttcatc atcggtcata aaatccgtat cctttacagg
     3901 atattttgca gtttcgtcaa ttgccgattg tatatccgat ttatatttat ttttcggtcg
     3961 aatcatttga acttttacat ttggatcata gtctaatttc attgcctttt tccaaaattg
     4021 aatccattgt ttttgattca cgtagttttc tgtattctta aaataagttg gttccacaca
     4081 taccaataca tgcatgtgct gattataaga attatcttta ttatttattg tcacttccgt
     4141 tgcacgcata aaaccaacaa gatttttatt aattttttta tattgcatca ttcggcgaaa
     4201 tccttgagcc atatctgaca aactcttatt taattcttcg ccatcataaa catttttaac
     4261 tgttaatgtg agaaacaacc aacgaactgt tggcttttgt ttaataactt cagcaacaac
     4321 cttttgtgac tgaatgccat gtttcattgc tctcctccag ttgcacattg gacaaagcct
     4381 ggatttacaa aaccacactc gatacaactt tctttcgcct gtttcacgat tttgtttata
     4441 ctctaatatt tcagcacaat cttttactct ttcagccttt ttaaattcaa gaatatgcag
     4501 aagttcaaag taatcaacat tagcgatttt cttttctctc catggtctca cttttccact
     4561 ttttgtcttg tccactaaaa cccttgattt ttcatctgaa taaatgctac tattaggaca
     4621 cataatatta aaagaaaccc ccatctattt agttatttgt ttggtcactt ataactttaa
     4681 cagatggggt ttttctgtgc aaccaatttt aagggttttc aatactttaa aacacataca
     4741 taccaacact tcaacgcacc tttcagcaac taaaataaaa atgacgttat ttctatatgt
     4801 atcaagataa gaaagaacaa gttcaaaacc atcaaaaaaa gacacctttt caggtgcttt
     4861 ttttatttta taaactcatt ccctgatctc gacttcgttc tttttttacc tctcggttat
     4921 gagttagttc aaattcgttc tttttaggtt ctaaatcgtg tttttcttgg aattgtgctg
     4981 ttttatcctt taccttgtct acaaacccct taaaaacgtt tttaaaggct tttaagccgt
     5041 ctgtacgttc cttaaggaat tctcatgttt gacagcttat catcgataag ctttaatgcg
     5101 gtagtttatc acagttaaat tgctaacgca gtcaggcacc gtgtatgaaa tctaacaatg
     5161 cgctcatcgt catcctcggc accgtcaccc tggatgctgt aggcataggc ttggttatgc
     5221 cggtactgcc gggcctcttg cgggatatcg tccattccga cagcatcgcc agtcactatg
     5281 gcgtgctgct agcgctatat gcgttgatgc aatttctatg cgcacccgtt ctcggagcac
     5341 tgtccgaccg ctttggccgc cgcccagtcc tgctcgcttc gctacttgga gccactatcg
     5401 actacgcgat catggcgacc acacccgtcc tgtggatctg acgcgtgtaa ctgcgaaggt
     5461 aaaacgggtg attgaaaata aactaacaaa tgaagaggaa cgaaagagaa gtctagaatt
     5521 tgttacgttt atatcggatg aattactgca aaatgatacg gagcctcgca catttattat
     5581 tcagaactct cttttatcgc tagagaaaat ccatactctc gcacagacca aagaggtaga
     5641 ggaatataaa gaagtcataa ctactctgac aagacatgat tcataatttg aaaagcaggt
     5701 aaactaaccc aaaaggcaag actctccgta gtttagaaaa gagcaggttg ctaataacat
     5761 ataaacagcc agttgccgtt atgataggtg actggctgca tgggatgaaa aggtgagggt
     5821 ggagacagac ataacactct taatagaaga gggtaattct ttctctttta tagaaaatca
     5881 attaattgaa agtagctcct tcattcttaa gatcaacgtg atataggttt gctaaccttt
     5941 gcgttcactt aactaactta taggggtaac acttaaaaaa gaatcaataa cgatagaaac
     6001 cgctcctaaa gcaggtgcat tttttcctaa cgaagaaggc aatagttcac atttattgtc
     6061 taaatgagaa tggactctag aagaaacttc gtttttaatc gtatttaaaa caatgggatg
     6121 agattcaatt atatgatttc tcaagataac agcttctata tcaaatgtat taaggatatt
     6181 ggttaatcca attccgatat aaaagccaaa gttttgaagt gcatttaaca tttctacatc
     6241 atttttattt gcgcgttcca caatctcttt tcgagaaata ttcttttctt ctttagagag
     6301 cgaagccagt aacgcttttt cagaagcata taattcccaa cagcctcgat ttccacagct
     6361 gcatttgggt ccattaaaat ctatcgtcat atgacccatt tccccagaaa aaccctgaac
     6421 acctttatac aattcgttgt taataacaag tccagttcca attccgatat taatactgat
     6481 gtaaacgatg ttttcatagt tttttgtcat accaaatact ttttcaccgt atgctcctgc
     6541 attagcttca ttttcaacaa aaaccggaac attaaactca ctctcaatta aaaactgcaa
     6601 atctttgata ttccaattta agttaggcat gaaaataatt tgctgatgac gatctacaag
     6661 gcctggaaca caaattccta ttccgactag accataaggg gactcaggca tatgggttac
     6721 aaaaccatga ataagtgcaa ataaaatctc ttttacttca ctagcggaag aactagacaa
     6781 gtcagaagtc ttctcgagaa taatatttcc ttctaagtcg gttagaattc cgttaagata
     6841 gtcgactcct atatcaatac caatcgagta gcctgcattc ttattaaaaa caagcattac
     6901 aggtcttctg ccgcctctag attgccctgc cccaatttca aaaataaaat ctttttcaag
     6961 cagtgtattt acttgagagg agacagtaga cttgtttaat cctgtaatct cagagagagt
     7021 tgccctggag acaggggagt tcttcaaaat ttcatctaat attaattttt gattcatttt
     7081 ttttactaaa gcttgatctg caatttgaat aataaccact cctttgttta tccaccgaac
     7141 taagttggtg ttttttgaag cttgaattag atatttaaaa gtatcatatc taatattata
     7201 actaaatttt ctaaaaaaaa cattgaaata aacatttatt ttgtatatga tgagataaag
     7261 ttagtttatt ggataaacaa actaactcaa ttaagatagt tgatggataa acttgttcac
     7321 ttaaatcaaa gggggaaatg tacaatgaaa aaagtactta tggcattcat tatttgttta
     7381 tcgctgattc tatctgtttt agccgctccg ccgtctggcg caggcgccgc attgaagatc
     7441 tccggagctc ccgggatccg gtaccggccg catg
//

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