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pET27b(+) 构建费用200元/个起

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基本信息

别名:

pET27b(+)

载体类型:

大肠杆菌蛋白表达

载体抗性:

卡那霉素 

载体标签:

N-pelB, C-His, C-HSV

筛选标记:

/

启动子:

T7, Lac

高拷贝/低拷贝:

载体大小:

5414bp

克隆方法:

多克隆位点,限制性内切酶

克隆菌株:

DH5ɑ

5'测序引物:

5'-TAATACGACTCACTATAGGG-3'

3'测序引物:

根据载体序列设计引物

组成型/诱导型:

诱导型

备注:

含有pelB信号肽,能够将目的蛋白定位于细胞周质腔,除抗性不同外,其它方面均与pET-25b相同。

质粒图谱和多克隆位点信息

注意:

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如要使用本公司保存的母源载体时,本公司不能保证所有母源载体完全符合客户预期,也不承担由此产生的任何责任


载体描述

The pET-27b(+) vector (Cat. No. 69863-3) carries an N-terminal pelB signal sequence for potential periplasmic localization, plus optional C-terminal HSV•Tag® and His•Tag® sequences. This vector differs from pET-25b(+) only by its selectable marker (kanamycin vs. ampicillin resistance).Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circular map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore, single-stranded sequencing should be performed using the T7 terminator primer (Cat. No. 69337-3).

 

载体序列

LOCUS       Exported                5414 bp ds-DNA     circular SYN 15-SEP-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    pET27b(+)

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 5414)

  AUTHORS   .

  TITLE     Direct Submission

  JOURNAL   Exported Sep 15, 2019 from SnapGene Viewer 4.2.6

            http://www.snapgene.com

FEATURES             Location/Qualifiers

     source          1..5414

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     terminator      26..73

                     /label=T7 terminator

                     /note="transcription terminator for bacteriophage T7 RNA

                     polymerase"

     CDS             complement(140..157)

                     /codon_start=1

                     /product="6xHis affinity tag"

                     /label=6xHis

                     /translation="HHHHHH"

     CDS             complement(164..196)

                     /codon_start=1

                     /product="HSV (herpes simplex virus) epitope tag"

                     /label=HSV tag

                     /translation="QPELAPEDPED"

     sig_peptide     complement(278..343)

                     /gene="pelB (fragment)"

                     /product="leader peptide for secretion"

                     /label=pelB signal sequence

     RBS             351..373

                     /note="efficient ribosome binding site from bacteriophage

                     T7 gene 10 (Olins and Rangwala, 1989)"

     protein_bind    388..412

                     /label=lac operator

                     /bound_moiety="lac repressor encoded by 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)."

     promoter        complement(413..431)

                     /label=T7 promoter

                     /note="promoter for bacteriophage T7 RNA polymerase"

     promoter        740..817

                     /gene="lacI"

                     /label=lacI promoter

     CDS             818..1900

                     /codon_start=1

                     /gene="lacI"

                     /product="lac repressor"

                     /label=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"

     CDS             2709..2900

                     /codon_start=1

                     /gene="rop"

                     /product="Rop protein, which maintains plasmids at low copy

                     number"

                     /label=rop

                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA

                     DELYRSCLARFGDDGENL"

     misc_feature    3002..3144

                     /label=bom

                     /note="basis of mobility region from pBR322"

     rep_origin      complement(3330..3918)

                     /direction=LEFT

                     /label=ori

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of

                     replication"

     CDS             4040..4855

                     /codon_start=1

                     /gene="aph(3')-Ia"

                     /product="aminoglycoside phosphotransferase"

                     /label=KanR

                     /note="confers resistance to kanamycin in bacteria or G418

                     (Geneticin(R)) in eukaryotes"

                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG

                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK

                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA

                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI

                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"

     rep_origin      complement(4948..5403)

                     /direction=LEFT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow

                     indicates direction of (+) strand synthesis"

ORIGIN

        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa

       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt

      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gacatcctcg gggtcttccg

      181 gggcgagttc tggctggcta gcccgtttga tctcgagtgc ggccgcaagc ttgtcgacgg

      241 agctcgaatt cggatccgaa ttaattccga tatccatggc catcgccggc tgggcagcga

      301 ggagcagcag accagcagca gcggtcggca gcaggtattt catatgtata tctccttctt

      361 aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt

      421 gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct acgccggacg catcgtggcc

      481 ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg

      541 gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca

      601 ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg

      661 gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag

      721 ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg

      781 atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga

      841 tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag

      901 ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat

      961 tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac

     1021 ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga

     1081 tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa

     1141 agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct

     1201 ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct

     1261 tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg

     1321 actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc

     1381 attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa

     1441 tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca

     1501 aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca

     1561 gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat

     1621 ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac

     1681 catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc

     1741 tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac

     1801 caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca

     1861 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa

     1921 gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag

     1981 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct

     2041 ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc

     2101 gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg

     2161 ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca

     2221 ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc

     2281 ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa

     2341 cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg

     2401 gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc

     2461 ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga

     2521 agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt

     2581 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga

     2641 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg

     2701 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc

     2761 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct

     2821 gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg

     2881 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag

     2941 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg

     3001 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc

     3061 atcagagcag attgtactga gagtgcacca tatatgcggt gtgaaatacc gcacagatgc

     3121 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc

     3181 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc

     3241 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg

     3301 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat

     3361 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag

     3421 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga

     3481 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg

     3541 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt

     3601 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac

     3661 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc

     3721 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt

     3781 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc

     3841 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc

     3901 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg

     3961 aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa

     4021 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg

     4081 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg

     4141 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct

     4201 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg

     4261 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc

     4321 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc

     4381 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat

     4441 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc

     4501 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc

     4561 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt

     4621 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg

     4681 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa

     4741 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga

     4801 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt

     4861 aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc

     4921 gcacatttcc ccgaaaagtg ccacctgaaa ttgtaaacgt taatattttg ttaaaattcg

     4981 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc

     5041 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga

     5101 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg

     5161 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag

     5221 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga

     5281 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg

     5341 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg

     5401 cgtcccattc gcca

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