| 質粒名稱: | pRSET a sfGCaMP6s-T78H |
|---|---|
| 出品公司: | Addgene |
| 目錄編號: | 55209 |
| 存儲實驗室: | Wolf Frommer |
|
載體骨架基本信息 |
|
| 載體名稱: | pRSET A |
| 載體出品公司: | Thermo Fisher Scientific |
| 空載體大小: | 2973bp |
| 完整質粒大小: | 4218 bp |
| 載體類型: | 細菌表達 |
| 載體抗性: | 氨芐霉素 |
| 生長溫度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷貝數: | 高拷貝 |
|
插入基因信息 |
|
| 插入基因名稱: | sfGCaMP6s-T78H |
| 插入大小: | 1245bp |
| 基因突變: | cpEGFP replaced with superfolder cpGFP and T78H mutation reintroduced |
| 啟動子: | T7 |
| 標簽/融合蛋白: |
6x HIS tag (N terminal on backbone)
Xpress_EK tag (N terminal on backbone)
|
| 克隆方法: | Ligation Independent Cloning |
| 3’克隆位點: | NotI (未破壞) |
| 5’測序引物 | T7 |
| 3’測序引物 | T7 term |
|
文獻引用方法 |
|
| 材料和方法部分: | pRSET a sfGCaMP6s-T78H was a gift from Wolf Frommer (Addgene plasmid # 100023 ; //n2t.net/addgene:100023 ; RRID:Addgene_100023) |
| 參考文獻部分: | Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins. Ast C, Foret J, Oltrogge LM, De Michele R, Kleist TJ, Ho CH, Frommer WB. Nat Commun. 2017 Sep 5;8(1):431. doi: 10.1038/s41467-017-00400-2. 10.1038/s41467-017-00400-2 [pii] PubMed 28874729 |
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pRSET a sfGCaMP6s-T78H載體質粒基本信息
pRSET a sfGCaMP6s-T78H質粒圖譜載體圖譜和 pRSET a sfGCaMP6s-T78H載體序列質粒序列多克隆位點信息
pRSET a sfGCaMP6s-T78H質粒載體簡介
pRSET a sfGCaMP6s-T78H質粒序列載體序列
LOCUS pRSET a sfGCaMP6s-T78H 4218 bp DNA circular SYN 15-APR-2025
DEFINITION Bacterial expression of fluorescent reporter for calcium signaling,
based off of GCaMP6s. cpEGFP replaced with a superfolder cpGFP
variant containing T78H mutation.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4218)
AUTHORS Ast C, Foret J, Oltrogge LM, De Michele R, Kleist TJ, Ho CH, Frommer
WB
TITLE Ratiometric Matryoshka biosensors from a nested cassette of green-
and orange-emitting fluorescent proteins.
JOURNAL Nat Commun. 2017 Sep 5;8(1):431. doi: 10.1038/s41467-017-00400-2.
PUBMED 28874729
REFERENCE 2 (bases 1 to 4218)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 15, 2025 from SnapGene Server 8.0.1
//gt12365.cn
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Commun."; date: "2017-09-5"; volume: "8(1):431. doi"; pages: "
10.1038/s41467-017-00400-2"
COMMENT Sequence Label: pRSET a sfGCaMP6s-T78H
FEATURES Location/Qualifiers
source 1..4218
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 1..20
/label=T7
/note="T7 promoter, forward primer"
promoter 1..19
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 51..73
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 93..110
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 114..146
/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 join(150..158,159..173)
/codon_start=1
/product="Xpress(TM) epitope tag, including an enterokinase
recognition and cleavage site"
/label=Xpress(TM) tag
/translation="DLYDDDDK"
regulatory 180..189
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 201..257
/codon_start=1
/product="calmodulin-binding peptide from avian smooth
muscle myosin light chain kinase"
/label=calmodulin-binding peptide
/translation="RRKWNKTGHAVRAIGRLSS"
primer_bind complement(1600..1618)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 1614..1661
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 1758..2213
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(1845..1864)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2055..2076
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 2239..2343
/gene="bla"
/label=AmpR promoter
CDS join(2344..2412,2413..3204)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(2562..2581)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 3375..3963
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 3864..3883
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4117..4134
/label=L4440
/note="L4440 vector, forward primer"
ORIGIN
1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
61 tttaagaagg agatatacat atgcggggtt ctcatcatca tcatcatcat ggtatggcta
121 gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatctcg
181 ccaccatggt cgactcatca cgtcgtaagt ggaataagac aggtcacgca gtcagagcta
241 taggtcggct gagctcactc gagaacgtgt atattaccgc ggataaacag aaaaacggca
301 ttaaagcgaa ctttcacgtg cgccataacg tggaagatgg cagcgtgcag ctggcggatc
361 attatcagca gaacaccccg attggcgatg gcccggtgct gctgccggat aaccattatc
421 tgagcaccca gaccaagctg agcaaagatc cgaacgaaaa acgcgatcac atggtgctgc
481 tggaatttgt gaccgcagcg ggcattacac acggcatgga tgaactgtat ggcggcaccg
541 gcggcagcgc gagccagggc gaagaactgt ttaccggcgt ggtgccgatt ctggtggaac
601 tggatggcga tgtgaacggc cataaattta gcgtgcgcgg cgaaggcgaa ggcgatgcga
661 ccattggcaa actgaccctg aaatttattt ccaccaccgg caaactaccg gtgccgtggc
721 cgaccctggt gaccacctta acctatggcg tgcagtgctt tagccgctat ccggatcata
781 tgaaacgcca tgattttttt aaaagcgcga tgccggaagg ctatgtgcag gaacgcacca
841 ttagctttaa agatgatggc aaatataaaa cccgcgcggt ggtgaaattt gaaggcgata
901 ccctggtgaa ccgcattgaa ctgaaaggca ccgattttaa agaagatggc aacattctgg
961 ggcataaact ggaatataac ctgccggacc aactgactga agagcagatc gcagaattta
1021 aagaggcttt ctccctattt gacaaggacg gggatgggac aataacaacc aaggagctgg
1081 ggacggtgat gcggtctctg gggcagaacc ccacagaagc agagctgcag gacatgatca
1141 atgaagtaga tgccgacggt gacggcacaa tcgacttccc tgagttcctg acaatgatgg
1201 caagaaaaat gaaatacagg gacacggaag aagaaattag agaagcgttc ggtgtgtttg
1261 ataaggatgg caatggctac atcagtgcag cagagcttcg ccacgtgatg acaaaccttg
1321 gagagaagtt aacagatgaa gaggttgatg aaatgatcag ggaagcagac atcgatgggg
1381 atggtcaggt aaactacgaa gagtttgtac aaatgatgac agcgaagtga gcggccgcga
1441 ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
1501 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttcgaagctt gatccggctg
1561 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
1621 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
1681 ccggatctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
1741 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
1801 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
1861 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
1921 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
1981 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
2041 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
2101 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
2161 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
2221 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
2281 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
2341 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
2401 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
2461 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
2521 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
2581 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
2641 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
2701 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
2761 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
2821 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
2881 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
2941 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
3001 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
3061 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
3121 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
3181 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
3241 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
3301 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
3361 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
3421 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
3481 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
3541 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
3601 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
3661 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
3721 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
3781 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
3841 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
3901 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
3961 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
4021 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
4081 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
4141 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagg
4201 atctcgatcc cgcgaaat
//
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