| 质粒名称: | SpyTag003-mKate2 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 133452 |
| 存储实验室: | Mark Howarth |
|
载体骨架基本信息 |
|
| 载体名称: | pET28a |
| 载体出品公司: | Clontech |
| 空载体大小: | 5369bp |
| 完整质粒大小: | 6024bp |
| 载体类型: | 细菌表达 |
| 载体抗性: | Kanamycin |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | SpyTag003-mKate2 |
| 物种来源: | Synthetic |
| 插入大小: | 807bp |
| 启动子: | T7 |
| 标签/融合蛋白: |
GSGSGS linker (C terminal on insert)
His6 (C terminal on insert)
|
| 克隆方法: | Gibson Cloning |
| 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: | SpyTag003-mKate2 was a gift from Mark Howarth (Addgene plasmid # 133452 ; http://n2t.net/addgene:133452 ; RRID:Addgene_133452) |
| 参考文献部分: | Approaching infinite affinity through engineering of peptide-protein interaction. Keeble AH, Turkki P, Stokes S, Khairil Anuar INA, Rahikainen R, Hytonen VP, Howarth M. Proc Natl Acad Sci U S A. 2019 Dec 10. pii: 1909653116. doi: 10.1073/pnas.1909653116. 10.1073/pnas.1909653116 PubMed 31822621 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS SpyTag003-mKate2 6024 bp ds-DNA
circular SYN 12-MAY-2021
DEFINITION Expresses SpyTag003-mKate2 (a far-red fluorescent
protein) in
bacterial cytoplasm.
ACCESSION .
VERSION .
KEYWORDS SpyTag003-mKate2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6024)
AUTHORS Keeble AH, Turkki P, Stokes S, Khairil Anuar INA,
Rahikainen R,
Hytonen VP, Howarth M
TITLE Approaching infinite affinity through engineering of
peptide-protein
interaction.
JOURNAL Proc Natl Acad Sci U S A. 2019 Dec 10. pii:
1909653116. doi:
10.1073/pnas.1909653116.
PUBMED 31822621
REFERENCE 2 (bases 1 to 6024)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6024
/organism="synthetic DNA construct"
/mol_type="other DNA"
RBS 15..37
/note="efficient ribosome binding site from
bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 117..809
/codon_start=1
/product="monomeric far-red fluorescent
protein (Shcherbo
et al., 2009)"
/label=mKate2
/note="mammalian codon-optimized"
/translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
DLPSKLGHR"
CDS 828..845
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(915..933)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 929..976
/label=T7 terminator
/note="transcription terminator for
bacteriophage T7 RNA
polymerase"
rep_origin 1013..1468
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of
replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(1100..1119)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 1310..1331
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(1560..2375)
/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 2283..2302
/label=Kan-R
/note="Kanamycin resistance gene, reverse
primer"
primer_bind 2367..2386
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 2497..3085
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC
origin of
replication"
primer_bind 2986..3005
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 3239..3256
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 3271..3410
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(3336..3355)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast
selectable
marker"
primer_bind 3496..3518
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(3512..3703)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains
plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(4512..5594)
/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 5556..5575
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(5595..5672)
/gene="lacI"
/label=lacI promoter
primer_bind 5878..5897
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream
of BamHI,
reverse primer"
primer_bind 5981..6000
/label=T7
/note="T7 promoter, forward primer"
promoter 5981..5999
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA
polymerase"
protein_bind 6000..6024
/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)."
ORIGIN
1 cctctagaaa taattttgtt taactttaag aaggagatat acatatggga
cgtggcgttc
61 ctcatattgt tatggtggac gcctacaaac gctataaatc gggaggtggt
tcaggcgtga
121 gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc
gtgaacaacc
181 accacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc
cagaccatga
241 gaatcaaggc cgtcgagggc ggccctctcc ccttcgcctt cgacatcctg
gctaccagct
301 tcatgtacgg cagcaaaacc ttcatcaacc acacccaggg catccccgac
ttctttaagc
361 agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac
gggggcgtgc
421 tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac
gtcaagatca
481 gaggggtgaa cttcccatcc aacggccctg tgatgcagaa gaaaacactc
ggctgggagg
541 cctccaccga gaccctgtac cccgctgacg gcggcctgga aggcagagcc
gacatggccc
601 tgaagctcgt gggcgggggc cacctgatct gcaacttgaa gaccacatac
agatccaaga
661 aacccgctaa gaacctcaag atgcccggcg tctactatgt ggacagaaga
ctggaaagaa
721 tcaaggaggc cgacaaagag acctacgtcg agcagcacga ggtggctgtg
gccagatact
781 gcgacctccc tagcaaactg gggcacagag gctccggctc cggctcccac
catcaccatc
841 accattgata agctagcacc actgagatcc ggctgctaac aaagcccgaa
aggaagctga
901 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
ctaaacgggt
961 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
ggacgcgccc
1021 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
cgctacactt
1081 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
cacgttcgcc
1141 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
tagtgcttta
1201 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
gccatcgccc
1261 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
tggactcttg
1321 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
ataagggatt
1381 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
taacgcgaat
1441 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
gtgcgcggaa
1501 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
aattaattct
1561 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
attatcaata
1621 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
gcagttccat
1681 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
aatacaacct
1741 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
agtgacgact
1801 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
aacaggccag
1861 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
tcgtgattgc
1921 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
aggaatcgaa
1981 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
atcaggatat
2041 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
ccatgcatca
2101 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
cagccagttt
2161 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
tttcagaaac
2221 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
ttgcccgaca
2281 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
taatcgcggc
2341 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
actgtttatg
2401 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
cgttccactg
2461 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
ttctgcgcgt
2521 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
tgccggatca
2581 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
taccaaatac
2641 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
caccgcctac
2701 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
agtcgtgtct
2761 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
gctgaacggg
2821 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
gatacctaca
2881 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
ggtatccggt
2941 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
acgcctggta
3001 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
tgtgatgctc
3061 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
ggttcctggc
3121 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
ctgtggataa
3181 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
ccgagcgcag
3241 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
ttacgcatct
3301 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
tgccgcatag
3361 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
ccccgacacc
3421 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
gcttacagac
3481 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
tcaccgaaac
3541 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
cagatgtctg
3601 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
tggcttctga
3661 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
tccgtgtaag
3721 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
ctcacgatac
3781 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
caactggcgg
3841 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
ttcgttaata
3901 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
cggaacataa
3961 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
ccgaagacca
4021 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
gttcgctcgc
4081 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
cgggtcctca
4141 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
atggcctgct
4201 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
gcgtgcaaga
4261 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
cggtcctcgc
4321 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
aagaagacag
4381 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
actgggttga
4441 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
cttacattaa
4501 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
ctgcattaat
4561 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
ggtttttctt
4621 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
agagagttgc
4681 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
ggtggttaac
4741 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
gatatccgca
4801 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
ctgatcgttg
4861 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
ttgttgaaaa
4921 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
attgcgagtg
4981 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
tgggcccgct
5041 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
tcgcgtaccg
5101 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
aagaaataac
5161 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
cagcggatag
5221 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
tttacaggct
5281 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
atcggcgcga
5341 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
ggtggcaacg
5401 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
aatgtaattc
5461 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
gctggcctgg
5521 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
atcgtataac
5581 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
tcatgccata
5641 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
ccttatgcga
5701 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
ccgccgcaag
5761 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
ctgccaccat
5821 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
ccccatcggt
5881 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
cggccacgat
5941 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
cactataggg
6001 gaattgtgag cggataacaa ttcc
//
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