| 质粒名称: | pET28a-sfGFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 85492 |
| 存储实验室: | Ryan Mehl |
|
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: | 5369 bp |
| 完整质粒大小: | 6150 bp |
| 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: | superfolder GFP,sfGFP |
| 物种来源: | 合成 |
| 插入大小: | 744 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | T7 |
| 标签/融合蛋白: | 6x His（插入件上的C端子） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | NcoI (未破坏) |
| 3' 克隆位点: | XhoI(未破坏) |
| 5' 测序引物 | T7 forward |
| 3' 测序引物 | T7 reverse |
|
文献引用方法 |
|
| 材料和方法部分: | pET28a-sfGFP was a gift from Ryan Mehl (Addgene plasmid # 85492 ; http://n2t.net/addgene:85492 ; RRID:Addgene_85492) |
| 参考文献部分: | Site-specific incorporation of unnatural amino acids as probes for protein conformational changes. Peeler JC, Mehl RA. Methods Mol Biol. 2012;794:125-34. doi: 10.1007/978-1-61779-331-8_8. 10.1007/978-1-61779-331-8_8 PubMed 21956560 |
买家导航
pET28asfGFP载体基本信息
pET28asfGFP载体图谱质粒图谱和多克隆位点信息
pET28asfGFP载体简介
pET28asfGFP载体序列
LOCUS pET28a-sfGFP 5975 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION pET28a expressing codon optimized superfolder GFP with C-terminal
His6 tag.
ACCESSION .
VERSION .
KEYWORDS pET28a-sfGFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5975)
AUTHORS Peeler JC, Mehl RA
TITLE Site-specific incorporation of unnatural amino acids as probes for
protein conformational changes.
JOURNAL Methods Mol Biol. 2012;794:125-34. doi: 10.1007/978-1-61779-331-8_8.
PUBMED 21956560
REFERENCE 2 (bases 1 to 5975)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..5975
/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=T7 Term
/note="T7 terminator, reverse primer"
CDS complement(140..157)
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS complement(169..186)
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
RBS 916..938
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind 953..977
/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(977..996)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(978..996)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(1080..1099)
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
promoter 1305..1382
/gene="lacI"
/label=lacI promoter
CDS 1383..2465
/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 complement(1402..1421)
/label=LacI-R
/note="LacI, reverse primer"
CDS 3274..3465
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
primer_bind complement(3459..3481)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
misc_feature 3567..3706
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 3622..3641
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind complement(3721..3738)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(3892..4480)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(3972..3991)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind complement(4591..4610)
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS 4602..5417
/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 complement(4675..4694)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
rep_origin complement(5509..5964)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(5646..5667)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 5858..5877
/label=F1ori-R
/note="F1 origin, reverse primer"
ORIGIN
1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagctttaat ggtgatgatg
181 atggtggctg cctttataca gttcatccat accgtgggta atgcccgcgg cggtaacaaa
241 ttccagcagc accatatgat cacgtttttc attcggatct ttgctcagaa cgctctgggt
301 gctcagataa tgattatccg gcagcagcac cgggccatca ccaatcgggg tattctgctg
361 ataatgatcc gccagctgca cgctaccatc ttccacgttg tgacggattt taaagttcgc
421 tttgatgcca tttttctgtt tatcggcggt aatatacaca ttatggctgt tgaaattata
481 ttccagttta tgacccagaa tgttgccatc ttctttaaaa tcaatacctt tcagttcaat
541 gcggttcacc agggtatcgc cttcaaattt aacttccgca cgggttttat aggtgccatc
601 atctttgaag ctaatggtac gttcctgcac atagccttcc ggcatcgcgc ttttaaagaa
661 atcatggcgt ttcatatgat ccggatagcg gctaaagcac tgaacgccat aggtcagggt
721 ggtcaccagg gtcggccacg gaaccggcag tttaccggtg gtgcaaataa atttcagggt
781 cagtttaccg ttggtcgcat caccttcgcc ttcgccacga acgctaaatt tatggccatt
841 cacatcacca tccagttcca ccagaatcgg cacaacgccg gtaaacagtt cttcaccttt
901 gctaaccatg gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt
961 tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat
1021 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
1081 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
1141 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
1201 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
1261 cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc
1321 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
1381 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
1441 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
1501 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
1561 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
1621 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
1681 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
1741 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
1801 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
1861 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
1921 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
1981 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
2041 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
2101 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
2161 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
2221 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
2281 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
2341 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
2401 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
2461 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg
2521 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc
2581 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg
2641 ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg
2701 cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc
2761 aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg
2821 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag
2881 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc
2941 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag
3001 tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt
3061 ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg
3121 tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt
3181 tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc
3241 atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt
3301 aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg
3361 gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac
3421 cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
3481 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
3541 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
3601 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg
3661 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
3721 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
3781 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
3841 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
3901 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
3961 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
4021 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
4081 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
4141 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
4201 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
4261 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
4321 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
4381 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
4441 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
4501 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa
4561 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
4621 aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
4681 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc
4741 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
4801 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
4861 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat
4921 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
4981 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
5041 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
5101 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
5161 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
5221 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
5281 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
5341 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
5401 tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg tatttagaaa
5461 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
5521 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
5581 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
5641 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
5701 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
5761 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
5821 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
5881 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
5941 ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
