請在線咨詢或直接聯系17720555249/18986294316

必騰預構載體定制最高100元/個

基本信息

質粒圖譜和多克隆位點信息

注意：

客戶在我公司選擇構建載體服務項目時，請自行提供母源載體。

如要使用本公司保存的母源載體時，本公司不能保證所有母源載體完全符合客戶預期，也不承擔由此產生的任何責任。

載體描述

pET-28a-Spy載體帶有一個N端的His/Spy/Thrombin/T7蛋白標簽，同時含有一個可以選擇的C端His標簽。注意：載體序列是以pBR322質粒的編碼規矩進行編碼的，所以T7蛋白表達區在質粒圖譜上面是反向的。

T7 RNA聚合酶啟動的克隆和表達區域在質粒圖譜中也被標注了出來。質粒的F1復制子是被定向的，所以在T7噬菌體聚合酶的作用下，包含有蛋白編碼序列的病毒粒子能夠產生，并啟動蛋白表達，同時蛋白表達將被T7終止子序列(Cat. No. 69337-3)的作用下終止蛋白翻譯。

載體序列

LOCUS       Exported                5444 bp ds-DNA     circular SYN 09-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    .

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 5444)

  AUTHORS   .

  TITLE     Direct Submission

  JOURNAL   Exported Thursday, Mar 14, 2019 from SnapGene Viewer 4.3.4

FEATURES             Location/Qualifiers

     source          1..5444

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     terminator      26..73

                     /label=T7 terminator

                     /note="transcription terminator for bacteriophage T7 RNA

                     polymerase"

     primer_bind     69..87

                     /label=T7t

     CDS             complement(140..157)

                     /codon_start=1

                     /product="6xHis affinity tag"

                     /label=6xHis

                     /translation="HHHHHH"

     misc_feature    158..197

                     /label=MCS

     CDS             complement(207..239)

                     /codon_start=1

                     /product="leader peptide from bacteriophage T7 gene 10"

                     /label=T7 tag (gene 10 leader)

                     /note="promotes efficient translation in E. coli"

                     /translation="MASMTGGQQMG"

     CDS             complement(243..260)

                     /codon_start=1

                     /product="thrombin recognition and cleavage site"

                     /label=thrombin site

                     /translation="LVPRGS"

     CDS             complement(270..287)

                     /codon_start=1

                     /product="6xHis affinity tag"

                     /label=6xHis

                     /translation="HHHHHH"

     misc_feature    300..371

                     /label=Spy

     RBS             381..403

                     /note="efficient ribosome binding site from bacteriophage

                     T7 gene 10 (Olins and Rangwala, 1989)"

     protein_bind    418..442

                     /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)."

     primer_bind     complement(442..461)

                     /label=T7

     promoter        complement(443..461)

                     /label=T7 promoter

                     /note="promoter for bacteriophage T7 RNA polymerase"

     promoter        770..847

                     /gene="lacI"

                     /label=lacI promoter

     CDS             848..1930

                     /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             2739..2930

                     /codon_start=1

                     /gene="rop"

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

                     number"

                     /label=rop

                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA

                     DELYRSCLARFGDDGENL"

     misc_feature    3032..3174

                     /label=bom

                     /note="basis of mobility region from pBR322"

     rep_origin      complement(3360..3948)

                     /direction=LEFT

                     /label=ori

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

                     replication"

     CDS             4070..4885

                     /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(4978..5433)

                     /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 gagtgcggcc gcaagcttgt

      181 cgacggagct cgaattcgga tccgcgaccc atttgctgtc caccagtcat gctagccata

      241 tggctgccgc gcggcaccag gccgctgctg tgatgatgat gatgatggct gctgcccatg

      301 gttcccgaac ctcctttggt cggtttatac gcatccacca tcacaatatg cgctgatccg

      361 gatccaccgc ccatggtata tctccttctt aaagttaaac aaaattattt ctagagggga

      421 attgttatcc gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc

      481 tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct

      541 ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg

      601 agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc

      661 atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg

      721 ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga

      781 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt

      841 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca

      901 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt

      961 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg

     1021 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca

     1081 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat

     1141 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg

     1201 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc

     1261 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag

     1321 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg

     1381 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct

     1441 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg

     1501 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt

     1561 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac

     1621 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga

     1681 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca

     1741 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct

     1801 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc

     1861 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag

     1921 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc

     1981 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga

     2041 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg

     2101 cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc

     2161 tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg

     2221 ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc

     2281 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt

     2341 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg

     2401 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc

     2461 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac

     2521 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc

     2581 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg

     2641 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta

     2701 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg

     2761 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg

     2821 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc

     2881 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc

     2941 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg

     3001 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata

     3061 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca

     3121 tatatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct

     3181 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca

     3241 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac

     3301 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt

     3361 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg

     3421 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc

     3481 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc

     3541 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc

     3601 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac

     3661 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt

     3721 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct

     3781 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc

     3841 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt

     3901 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg

     3961 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc

     4021 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat

     4081 tcaacgggaa acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata

     4141 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta

     4201 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga

     4261 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat

     4321 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa

     4381 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct

     4441 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga

     4501 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag

     4561 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa

     4621 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct

     4681 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga

     4741 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca

     4801 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca

     4861 tttgatgctc gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta

     4921 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaaa

     4981 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt

     5041 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag

     5101 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg

     5161 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat

     5221 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc

     5281 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga

     5341 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac

     5401 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gcca

//