| 质粒名称: | pNIC28-Bsa4 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 26103 |
| 存储实验室: | Opher Gileadi |
|
载体骨架基本信息 |
|
| 载体名称: | pET28a |
| 载体出品公司: | Novagen |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | 5353 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 低拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | -- |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | EF198106 |
| 启动子 | -- |
| 标签/融合蛋白: | His-TEV（主干上的N终端） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | 独立于连接的克隆（在克隆过程中销毁） |
| 3' 克隆位点: | 独立于连接的克隆（在克隆过程中销毁） |
| 5' 测序引物 | T7F |
| 3' 测序引物 | T7R |
|
文献引用方法 |
|
| 材料和方法部分: | pNIC28-Bsa4 was a gift from Opher Gileadi (Addgene plasmid # 26103 ; http://n2t.net/addgene:26103 ; RRID:Addgene_26103) |
| 参考文献部分: | High-throughput production of human proteins for crystallization: The SGC experience. Savitsky P, Bray J, Cooper CD, Marsden BD, Mahajan P, Burgess-Brown NA, Gileadi O. J Struct Biol. 2010 Jun 10. ():. 10.1016/j.jsb.2010.06.008 PubMed 20541610 |
买家导航
pNIC28Bsa4载体基本信息
pNIC28Bsa4载体图谱质粒图谱和多克隆位点信息
pNIC28Bsa4载体简介
pNIC28Bsa4载体序列
LOCUS pNIC28-Bsa4 7280 bp ds-DNA circular SYN 18-NOV-2019
DEFINITION SGC Empty backbone for bacterial expression.
ACCESSION .
VERSION .
KEYWORDS pNIC28-Bsa4
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7280)
AUTHORS Savitsky P, Bray J, Cooper CD, Marsden BD, Mahajan P, Burgess-Brown
NA, Gileadi O
TITLE High-throughput production of human proteins for crystallization:
The SGC experience.
JOURNAL J Struct Biol. 2010 Jun 10. ():.
PUBMED 20541610
REFERENCE 2 (bases 1 to 7280)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Nov 18, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7280
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 1..20
/label=T7
/note="T7 promoter, forward primer"
promoter 1..19
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 20..44
/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)."
RBS 59..81
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 92..109
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 134..154
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
promoter 170..615
/gene="sacR"
/label=sacB promoter
/note="sacB promoter and control region"
CDS 616..2037
/codon_start=1
/gene="Bacillus subtilis sacB"
/product="secreted levansucrase that renders bacterial
growth sensitive to sucrose"
/label=SacB
/note="negative selection marker"
/translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
VKDSILEQGQLTVNK"
CDS 2145..2162
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(2215..2233)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 2229..2276
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 2313..2768
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2400..2419)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2610..2631
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(2860..3675)
/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"
primer_bind 3583..3602
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 3667..3686
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 3797..4385
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4286..4305
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4539..4556
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 4571..4710
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(4636..4655)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4796..4818
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(4812..5003)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(5812..6894)
/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"
primer_bind 6856..6875
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(6895..6972)
/gene="lacI"
/label=lacI promoter
primer_bind 7178..7197
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
ORIGIN
1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
121 agatctgggt accgagaacc tgtacttcca atccatggag accgacgtcc acatatacct
181 gccgttcact attatttagt gaaatgagat attatgatat tttctgaatt gtgattaaaa
241 aggcaacttt atgcccatgc aacagaaact ataaaaaata cagagaatga aaagaaacag
301 atagattttt tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa
361 caaaagagga aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa
421 agtaaatcgc gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca
481 aagtgtatac tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact
541 aacttgccat cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa
601 aaggagacat gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt
661 actaccgcac tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag
721 ccatataagg aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct
781 gaacagcaaa aaaatgaaaa atataaagtt cctgagttcg attcgtccac aattaaaaat
841 atctcttctg caaaaggcct ggacgtttgg gacagctggc cattacaaaa cactgacggc
901 actgtcgcaa actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat
961 gcggatgaca catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc
1021 tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc
1081 ctaaaagacc aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc
1141 cgtttattct acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca
1201 caagttaacg tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa
1261 tcaatctttg acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc
1321 aactacagct caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc
1381 cacaaatact tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa
1441 tctttattta acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa
1501 aaacttctgc aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg
1561 attgagctaa acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac
1621 acagtaacag atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg
1681 ttcactgact cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac
1741 atgcttggtt atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc
1801 cttgtgttaa aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct
1861 gtacctcaag cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc
1921 tacgcagaca aacaatcaac gtttgcgcct agcttcctgc tgaacatcaa aggcaagaaa
1981 acatctgttg tcaaagacag catccttgaa caaggacaat taacagttaa caaataaaaa
2041 cgcaaaagaa aatgccgata tcctattggc attgacggtc tccagtaaag gtggatacgg
2101 atccgaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc
2161 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
2221 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
2281 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
2341 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
2401 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
2461 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
2521 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
2581 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
2641 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
2701 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
2761 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
2821 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
2881 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
2941 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
3001 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
3061 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
3121 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
3181 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
3241 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
3301 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
3361 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
3421 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
3481 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
3541 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
3601 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
3661 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
3721 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
3781 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
3841 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
3901 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
3961 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
4021 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
4081 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
4141 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
4201 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
4261 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
4321 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
4381 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
4441 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
4501 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
4561 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
4621 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
4681 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
4741 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
4801 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
4861 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
4921 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
4981 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
5041 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
5101 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
5161 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
5221 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
5281 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
5341 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
5401 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
5461 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
5521 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
5581 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
5641 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
5701 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
5761 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
5821 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
5881 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
5941 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
6001 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
6061 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
6121 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
6181 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
6241 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
6301 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
6361 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
6421 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
6481 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
6541 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
6601 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
6661 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
6721 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
6781 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
6841 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
6901 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
6961 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
7021 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
7081 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
7141 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
7201 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
7261 agatctcgat cccgcgaaat
//
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pNIC28Bsa4载体质粒应用举例
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