买家导航
pHIS1525载体质粒基本信息
pHIS1525质粒图谱载体图谱和pHIS1525载体序列质粒序列多克隆位点信息
pHIS1525质粒载体简介
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.
pHIS1525质粒序列载体序列
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
//
pHIS1525载体图谱质粒图谱pdf版和pHIS1525载体序列质粒序列等相关资料下载
pHIS1525质粒载体应用举例
- 上一篇：pHT43
- 下一篇：pK18mobSacB
