pLLP-STII/Plpp-STII大肠表达质粒

基本信息

启动子:	lpp/lac
复制子:	ColE1 ori
质粒分类:	大肠杆菌载体；PET系列表达质粒
质粒大小:	7559bp
质粒标签:	N-STII，C-6×His
原核抗性:	氨苄青霉素Amp
克隆菌株:	DH5α
培养条件:	37℃，有氧，LB
表达宿主:	BL21(DE3)
培养条件:	37℃，有氧，LB
诱导方式:	IPTG或乳糖及其类似物
5'测序引物:	5 GGC TTT ACA CTT TAT GCT TC 3
3'测序引物:	5CCA TTT TTC ACT TCA CAG G 3

质粒简介

        pLLP-STII表达载体采用E.coli强启动lpp和STII分泌信号肽，同时在C端添加了6个His便于采用chelating sepharose亲和层析纯化目标蛋白。STII信号肽引导能力强且易于被E.coli信号肽酶切割。即使第一个氨基酸是半胱胺酸的干扰素也可以实现分泌表达。

质粒图谱

质粒序列

LOCUS       Exported                7559 bp ds-DNA     circular SYN 03-AUG-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pLLP-STII
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7559)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, September 6, 2016 from SnapGene Viewer 3.1.4
FEATURES             Location/Qualifiers
     source          1..7559
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        247..277
                     /note="lac UV5 promoter"
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    285..301
                     /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)."
     primer_bind     309..325
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     RBS             361..383
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    424..459
                     /note="STll signal"
     CDS             484..501
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     promoter        1866..1943
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             1944..3026
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="lacI"
                     /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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    3039..3060
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             5125..5316
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    5418..5558
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5744..6332)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(6503..7363)
                     /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(7364..7468)
                     /gene="bla"
                     /note="AmpR promoter"
ORIGIN
        1 ggataaccag aagcaataaa aaatcaaatc ggatttcact atataatctc actttatcta
       61 agatgaatcc gatggaagca tcctgttttc tctcaatttt tttatctaaa acccagcgtt
      121 cgatgcttct ttgagcgaac gatcaaaaat aagtgccttc ccatcaaaaa aatattctca
      181 acataaaaaa ctttgtgtaa tacttgtaac gctacatgga gattaactca atctagctag
      241 agaggcttta cactttatgc ttccggctcg tataatgtgt ggaattgtga gcggataaca
      301 atttcacaca ggaaacagct atgaccatga ttacggattc actggaactc tagaaataat
      361 tttgtttaac tttaagaagg agatatacat atgaaaaaga acatcgcatt cctcctggca
      421 tctatgtttg ttttctctat cgctaccaac gcttacgctg gatccgcaga attcagcgct
      481 agccaccatc atcatcatca ttaagcttag atccggctga gcaacgacgt gaacgcaatg
      541 cgttccgacg ttcaggctgc taaagatgac gcagctcgtg ctaaccagcg tctggacaac
      601 atggctacta aataccgcaa gtaatagtac ctgtgaagtg aaaaatggcg cacattgtgc
      661 gacatttttt ttgtctgccg tttaccgcta ctgcgtcacg cgtaacatat tcccttgctc
      721 tggttcacca ttctgcgctg actctactga aggcgcattg ctggctgcgg gagttgctcc
      781 actgctcacc gaaaccggat accctgcccg acgatacaac gctttatcga ctaacttctg
      841 atctacagcc ttattgtctt taaattgcgt aaagcctgct ggcagtgtgt atggcattgt
      901 ctgaacgttc tgctgttctt ctgccgatag tggtcgatgt acttcaacat aacgcatccc
      961 gttaggctcc acggaatatt tcaccggttc gttgatcact ttcaccggcg ttcccgtccg
     1021 cacgctggag aacaaggctt taatatccgg tgcattcatg cgaatacacc ctgaactgac
     1081 gcgcaaaccg acgctgtccg gcgcactggt accatgaatg aggtattcgc cattaccatg
     1141 cgcgaggcgc agtgcgtaac gtcctagcgg gttatttggt ccggcaggaa cgactggcgg
     1201 taatttaatg ccacgctcca gcgaacgctg acgaatgcct gccgtaggcg tccaggttgg
     1261 gttagggatt ttctgcccaa cacgcgtttc catcaccggc gtttccagcc cctgcaatcc
     1321 aatacctatt ggataaacct gcacaatatt ttctcccggc ggataataat aaaggcgcag
     1381 ctctgcaagg ttggtcgacg gtcgaccggt ctcacttcaa cataacgcat cccgttaggc
     1441 tccacggaat atttcaccgg ttcgttgatc actttcaccg gcgttcccgt ccgcacgctg
     1501 gagaacaagg ctttaatatc cggtgcattc atgcgaatac accctgaact gacgcgcaaa
     1561 ccgacgctgt ccggcgcact ggtaccatga atgaggtatt cgccattacc atgcgcgagg
     1621 cgcagtgcgt aacgtcctag cgggttattt ggtccggcag gaacgactgg cggtaattta
     1681 atgccacgct ccagcgaacg ctgacgaatg cctgccgtag gcgtccaggt tgggttaggg
     1741 attttctgcc caacacgcgt ttccatcacc ggcgtttcca gcccctgcaa tccaatacct
     1801 attggataaa cctgcacaat attttctccc ggcggataat aataaaggcg cagctctgca
     1861 aggttgacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     1921 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     1981 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     2041 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     2101 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     2161 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     2221 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     2281 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     2341 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     2401 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     2461 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     2521 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     2581 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     2641 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     2701 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
     2761 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     2821 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     2881 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     2941 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     3001 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     3061 taggcacccc aggctttaca ctttatgcta acgataatcc ctgacgcggt gcatcaggta
     3121 ataacagttg tgaaggaata gttatcgtcg taccaggttt tggcaccggg gcgatagtgt
     3181 tattggcttc aaggatcaac attgccgcag tatcaaaacg tcgggcaata gcctgaaggt
     3241 ttttatcccc ttcttgcacc gtatacgttt gattttgccc aaccagtcgg cttccggttg
     3301 gtggtagcgg ataatcaacc gcccaggcag cctggatggc gctaaaagcg ccgataagcg
     3361 tgagtgtaag caaagacgcg cgtttcattg taaacctcct gtatttgccg gagactcacg
     3421 ctgaaacgtc ggatggcgct tatgttcacc tgaaaccaaa acactcctgt gcaggtcagt
     3481 gtaaacattg accatccggc aatgtgagcc aaccggatga aagctgtcct tttagtttag
     3541 ctaagtgcag cggctttggc gcgaattgcg cgaatcatcg cttccagacc ttgtgaacga
     3601 gatggggtga gatgttgggt gagcgccatt ttttcaaacc acggacgcac atcgaaattg
     3661 acaatatcct gcggcgtcat ctgatcgtag agaataaaga cgaccgcaat aagccctttc
     3721 acaatcgccg catcgctgtc gccctgtaat tcaataattc cctgggcatt ctggcgcatg
     3781 acaatccaca cctgactctg acagccctga atgctatttt gtggacttct gtcttcgtcg
     3841 cgtaattctg gcagacgctg ggggaccgat gcccttgaga gccttcaacc cagtcagctc
     3901 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     3961 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     4021 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     4081 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     4141 cggcatggcg gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc gaggctggat
     4201 ggccttcccc attatgattc ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc
     4261 catgctgtcc aggcaggtag atgacgacca tcagggacag cttcaaggat cgctcgcggc
     4321 tcttaccagc ctaacttcga tcactggacc gctgatcgtc acggcgattt atgccgcctc
     4381 ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc gccctatacc ttgtctgcct
     4441 ccccgcgttg cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg
     4501 cacctcgcta acggattcac cactccaaga attggagcca atcaattctt gcggagaact
     4561 gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg cgtccgccat ctccagcagc
     4621 cgcacgcggc gcatctcggg cagcgttggg tcctggccac gggtgcgcat gatcgtgctc
     4681 ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca
     4741 ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca
     4801 acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc
     4861 tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct
     4921 acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc
     4981 atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca
     5041 gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga
     5101 aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc
     5161 cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat
     5221 gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc
     5281 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     5341 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     5401 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
     5461 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     5521 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     5581 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5641 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5701 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5761 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5821 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5881 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5941 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6001 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6061 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6121 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6181 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6241 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6301 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6361 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6421 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6481 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6541 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6601 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6661 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6721 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6781 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
     6841 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6901 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6961 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7021 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7081 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac
     7141 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7201 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7261 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7321 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7381 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7441 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     7501 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggcccttt