| 质粒名称: | pET28:GFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 60733 |
| 存储实验室: | Matthew Bennett |
|
载体骨架基本信息 |
|
| 载体名称: | pET-28b |
| 载体出品公司: | Clontech |
| 空载体大小: | 5273 bp |
| 完整质粒大小: | 5993 bp |
| 载体类型: | Bacterial Expression, Synthetic Biology |
| 载体抗性: | Kanamycin, 50 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH10B |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | GFP |
| 插入大小: | 720bp |
|
文献引用方法 |
|
| 材料和方法部分: | pET28:GFP was a gift from Matthew Bennett (Addgene plasmid # 60733 ; http://n2t.net/addgene:60733 ; RRID:Addgene_60733) |
| 参考文献部分: | Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants. Shis DL, Bennett MR. Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5028-33. doi: 10.1073/pnas.1220157110. Epub 2013 Mar 11. 10.1073/pnas.1220157110 PubMed 23479654 |
买家导航
pET28:GFP载体质粒基本信息
pET28:GFP质粒图谱载体图谱和pET28:GFP载体序列质粒序列多克隆位点信息
pET28:GFP质粒载体简介
pET28:GFP质粒序列载体序列
LOCUS pET28:GFP 5989 bp DNA circular SYN 14-APR-2025
DEFINITION pET28 driving expression of GFP, biobrick part Bba_E0040.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5989)
AUTHORS Shis DL, Bennett MR
TITLE Library of synthetic transcriptional AND gates built with split T7
RNA polymerase mutants.
JOURNAL Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5028-33. doi:
10.1073/pnas.1220157110. Epub 2013 Mar 11.
PUBMED 23479654
REFERENCE 2 (bases 1 to 5989)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 14, 2025 from SnapGene Server 8.0.1
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1073/pnas.1220157110"; journalName: "Proc Natl Acad Sci U S A";
date: "2013-03-26- 26"; volume: "110"; issue: "13"; pages: "5028-33"
COMMENT Sequence Label: pET28:GFP
FEATURES Location/Qualifiers
source 1..5989
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator complement(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(join(204..917,918..920,921..923))
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS complement(join(207..917,918..920,921..923))
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(249..270)
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind 598..617
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 857..878
/label=EGFP-N
/note="EGFP, reverse primer"
RBS 930..952
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind 967..991
/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(991..1010)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(992..1010)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(1094..1113)
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
promoter 1319..1396
/gene="lacI"
/label=lacI promoter
CDS 1397..2479
/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(1416..1435)
/label=LacI-R
/note="LacI, reverse primer"
CDS 3288..3479
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
primer_bind complement(3473..3495)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
misc_feature 3581..3720
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 3636..3655
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind complement(3735..3752)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(3906..4494)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(3986..4005)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind complement(4605..4624)
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS 4616..5431
/codon_start=1
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
CDS 4616..5431
/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(4689..4708)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
rep_origin complement(5523..5978)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(5660..5681)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 5872..5891
/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 gagtgcggcc gcaagcttgt
181 cgacggagct cgaattcgga tccttacttg tacagctcgt ccatgccgag agtgatcccg
241 gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc
301 agggcggact gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg
361 ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg
421 atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg
481 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
541 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
601 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg
661 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
721 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
781 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
841 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
901 agctcctcgc ccttgctcac catggtatat ctccttctta aagttaaaca aaattatttc
961 tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg
1021 atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
1081 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc
1141 gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg
1201 ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac
1261 ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga
1321 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
1381 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
1441 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
1501 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
1561 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
1621 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
1681 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
1741 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
1801 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
1861 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
1921 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
1981 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
2041 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
2101 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
2161 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
2221 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
2281 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
2341 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
2401 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
2461 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
2521 cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
2581 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
2641 aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga
2701 tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca
2761 ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc
2821 cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc
2881 ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca
2941 aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc
3001 tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct
3061 gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag
3121 tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca
3181 gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg
3241 gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg
3301 aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga
3361 aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg
3421 ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
3481 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
3541 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
3601 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
3661 agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
3721 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
3781 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
3841 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
3901 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
3961 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
4021 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
4081 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
4141 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
4201 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
4261 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
4321 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
4381 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
4441 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
4501 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
4561 ttggtcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
4621 ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga
4681 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
4741 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
4801 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
4861 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
4921 cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
4981 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
5041 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
5101 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
5161 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
5221 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
5281 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
5341 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
5401 gtttcatttg atgctcgatg agtttttcta agaattaatt catgagcgga tacatatttg
5461 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
5521 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
5581 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
5641 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
5701 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
5761 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
5821 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
5881 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
5941 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
