| 质粒名称: | pTEF:ATP |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 92179 |
| 存储实验室: | Matthias Heinemann |
|
载体骨架基本信息 |
|
| 载体名称: | HO-poly-KanMX4-HO |
| 载体出品公司: | Voth et al. (2001) Nucleic Acids Res (PMID: 11410682) |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 6062 bp |
| 完整质粒大小: | 6038 bp |
| 载体修饰: | 除poly区域外，整个主干被使用。因为我们用Gibson-DNA组装来构建质粒，所以我们不需要限制性位点和多聚区。 |
| 载体类型: | 酵母表达；整合和表达到酵母中。 |
| 筛选标记: | 新霉素（G418） |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | ATeam1.03 codon optimized for yeast (Saccharomyces cerevisiae),ATP FRET sensor |
| 物种来源: | Synthetic |
| 插入大小: | 1836 bp |
| 启动子 | TEF1p |
| 标签/融合蛋白: | 酵母密码子优化mseCFP（供体）和cp173 mVenus（受体）是ATeam1.03 FRET传感器的一部分。 |
| 克隆方法: | Gibson克隆 |
| 5' 测序引物 | GGAGTATTGTGTCATGTTCG |
| 3' 测序引物 | GACAGTCACATCATGCC |
|
文献引用方法 |
|
| 材料和方法部分: | pTEF:ATP was a gift from Matthias Heinemann (Addgene plasmid # 92179 ; http://n2t.net/addgene:92179 ; RRID:Addgene_92179) |
| 参考文献部分: | Autonomous Metabolic Oscillations Robustly Gate the Early and Late Cell Cycle. Papagiannakis A, Niebel B, Wit EC, Heinemann M. Mol Cell. 2017 Jan 19;65(2):285-295. doi: 10.1016/j.molcel.2016.11.018. Epub 2016 Dec 15. 10.1016/j.molcel.2016.11.018 PubMed 27989441 |
买家导航
pTEFATP载体基本信息
pTEFATP载体图谱质粒图谱和多克隆位点信息
pTEFATP载体简介
pTEFATP载体序列
LOCUS pTEF:ATP 8554 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION HO-TEF1p-ATeam1.03-KanMX4-HO. Codon optimized for yeast version of
the ATeam1.03 ATP FRET sensor (originally from Imamura et al. PNAS,
2009 ), expressed via the TEF1 promoter. HO locus integration..
ACCESSION .
VERSION .
KEYWORDS pTEF:ATP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8554)
AUTHORS Papagiannakis A, Niebel B, Wit EC, Heinemann M
TITLE Autonomous Metabolic Oscillations Robustly Gate the Early and Late
Cell Cycle.
JOURNAL Mol Cell. 2017 Jan 19;65(2):285-295. doi:
10.1016/j.molcel.2016.11.018. Epub 2016 Dec 15.
PUBMED 27989441
REFERENCE 2 (bases 1 to 8554)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8554
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 97..118
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
protein_bind 173..189
/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 178..200
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 197..213
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 197..213
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
terminator 1205..1394
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
promoter complement(3280..3678)
/gene="S. cerevisiae TEF1"
/label=TEF1 promoter
/note="promoter for EF-1-alpha"
gene 3761..5117
/label=kanMX
/note="yeast selectable marker conferring kanamycin
resistance (Wach et al., 1994)"
promoter 3761..4104
/label=TEF promoter
/note="Ashbya gossypii TEF promoter"
CDS 4105..4914
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind complement(4172..4191)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
terminator 4920..5117
/label=TEF terminator
/note="Ashbya gossypii TEF terminator"
primer_bind complement(5725..5744)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(5726..5744)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(5751..5768)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(5751..5767)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5760..5782)
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind complement(6067..6086)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 6199..6220
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(6374..6393)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 6493..6515
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(6553..6571)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 6639..6743
/gene="bla"
/label=AmpR promoter
CDS 6744..7604
/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(6962..6981)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 7775..8363
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8264..8283
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 8517..8534
/label=L4440
/note="L4440 vector, forward primer"
ORIGIN
1 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
61 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
121 ggcaccccag gctttacact ttatgcttcc gcggctcgta tgttgtgtgg aattgtgagc
181 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccactag tccgaggcct
241 cgagatccga tatcgccgtg gcggccgcca gctgaagctt aattatcctg ggcacgagtg
301 aaacaaagct aaaaccttta tttagcatgg ccattgaatg taacaattat atatatcgca
361 agcacaaaaa atcaaggaga gagaactacc actttgttca tgtgtacaat gttcattatc
421 tccataagca aaaaaaaaaa aatagaaaac atatgctata aggttgatat tctcacgagt
481 aagcggcact tgctacttat tgacattgca gatttttggc tacagaaata gtatattaga
541 gattataatt gctaatcaaa tcaaaatata aaattagtaa accaaaccat ttataccctt
601 ccttagtagt tatggattgt tttttaatga tatttctgca aaccaaagaa agattgttat
661 ccagatagaa tttagttttg atattcattt ttttgttgaa gattgaacgc catatctggg
721 cctcataatt caaaagacgg tgccattatc ggtagcgttt cgcattgtac tggatttcag
781 aaatttcaca gttgatgaat cgaaaagaat ggtctcattg caacacgtaa ggttaagatg
841 tcccttttta ccattatagg caataaatga atcataaaac gaccgtatac tggtgaaata
901 gtagggagaa cgagtacctg tagtaaaaag tataaatcat agttaatcgg gcaatgtccc
961 tcgatcaagg agtattgtgt catgttcgag acaaacgcca acatttttgt ttcttttgga
1021 caaatgttgt ttgcatttat gatccgttat attttgatct aatgtagagt tgcacgtagt
1081 tcttactggc aaagaaatcg atgcatacca aaaaagaata aaggtgatat ttgatcttta
1141 ccgtttagtt ccaacgtaaa attgtgcctt tggacttaaa atggcgtcgt acgctgagct
1201 ggatcttcga gcgtcccaaa accttctcaa gcaaggtttt cagtataatg ttacatgcgt
1261 acacgcgtct gtacagaaaa aaaagaaaaa tttgaaatat aaataacgtt cttaatacta
1321 acataactat aaaaaaataa atagggacct agacttcagg ttgtctaact ccttcctttt
1381 cggttagagc ggatcttagc tagccgcggt accaagctta ctcgagtcat tcaatgttat
1441 gtctaatttt gaagttggcc ttgataccat ttttttgttt gtcagcagtg atgtagacgt
1501 tgtgagagtt ataattgtat tctaacttat gacctaaaat gttaccatct tctttaaaat
1561 caataccttt caattcaatt ctattgacca aagtgtcacc ttcaaatttt acttcagctc
1621 tggtcttgta attaccatcg tccttaaaga aaatagttct ttcttggaca taaccttcag
1681 gcattgcaga cttaaaaaag tcatgttgct tcatgtggtc aggatatcta gcgaagcatt
1741 gtaaaccgta acccaatgtt gtaactaatg taggccaagg aacaggtaac ttaccggtag
1801 tgcatatcaa tttcaaagtc aatttaccgt atgttgcgtc accttcacct tcaccactaa
1861 ctgaaaactt atgaccattg acgtcaccgt ctaattctac taaaataggg acgacacctg
1921 tgaataattc ttcaccttta ctaaccatac caccagaacc acccttgtat aattcatcca
1981 tacccaaagt aataccggca gcggtgacaa attctaataa aaccatatga tctctttttt
2041 cgttaggatc tttggacaac ttagattggt aggacaaata gtgattatca ggtaataata
2101 caggaccgtc accgataggt gtgttttgtt gatagtggtc tgctaattga acaccaccgt
2161 ccatgaattc gttagcctta ccagcaacat ccaatctgtt caaagctctt tgtaaagcca
2221 attctgctct tctgatatcg gtatcatcag attgagagtt caatctttct tgggctcttt
2281 gtctagcagc ttcagctctt tctttgtcaa taccttcagc tgtttcagca gcttgagcgt
2341 taatagtaac atgatctggt ctaacttcaa cagtaccacc tgaaactgca accatttctg
2401 tttgaccgtc ctttttcaat ctaacggcac caattttcaa tggggctttt gtaggaatat
2461 gacctggcaa aatacccaaa tcaccagatt cagctctgac agaaaccatt tcaatatcgg
2521 catcgtaaac aggaccatct ggagtagtaa tgttaacctt aacggtcttc atatcgatag
2581 cagcagtaac aaattccaac aagaccatgt gatctctttt ttcgtttggg tccttagaca
2641 atttggattg ggtagacaag taatggttat ctggcaacaa aactggacca tcaccaattg
2701 gagtgttttg ttggtaatga tcagccaatt gtacaccacc atcttcgatg ttgtgtctga
2761 tcttgaaatg agccttaata ccgttctttt gcttatcggc ggtaatgtaa acgttatggg
2821 agatgtagtt gtattccaac ttgtgaccca agatgttacc atcttctttg aagtcgatac
2881 ccttcaattc gattctgtta accaaggtat caccttcgaa cttaacttcg gctctagttt
2941 tgtagttacc gtcatccttg aagaagatgg ttctttcttg aacgtaacct tctggcatag
3001 cggatttgaa gaaatcgtgt tgcttcatat gatctgggta tctagcaaaa cattgaacac
3061 cccaagtcaa agtagtaacc aaagttggcc atggaactgg caatttacct gtggtacaga
3121 tgaacttcaa ggtcaattta ccataagtag catcaccttc accttcacca gaaacagaaa
3181 atctgtgacc gttaacatca ccatccaatt caaccaagat tggaacaaca ccagtgaata
3241 attcttcacc cttagaaacc attttaataa cctaggaaac ttagattaga ttgctatgct
3301 ttctttctaa tgagcaagaa gtaaaaaaag ttgtaataga acaagaaaaa tgaaactgaa
3361 acttgagaaa ttgaagaccg tttattaact taaatatcaa tgggaggtca tcgaaagaga
3421 aaaaaatcaa aaaaaaaatt ttcaagaaaa agaaacgtga taaaaatttt tattgccttt
3481 ttcgacgaag aaaaagaaac gaggcggtct cttttttctt ttccaaacct ttagtacggg
3541 taattaacga caccctagag gaagaaagag gggaaattta gtatgctgtg cttgggtgtt
3601 ttgaagtggt acggcgatgc gcggagtccg agaaaatctg gaagagtaaa aaaggagtag
3661 aaacattttg aagctatggt gtggaattcc agctgcgggt taattaaggc gcgccagatc
3721 tgtttagctt gcctcgtccc cgccgggtca cccggccagc gacatggagg cccagaatac
3781 cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat
3841 ttagcccata catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc
3901 gcgaagcaaa aattacggct cctcgctgca gacctgcgag cagggaaacg ctcccctcac
3961 agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt
4021 gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat acagttctca
4081 catcacatcc gaacataaac aaccatgggt aaggaaaaga ctcacgtttc gaggccgcga
4141 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
4201 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg
4261 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
4321 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
4381 tggttactca ccactgcgat ccccggcaaa acagcattcc aggtattaga agaatatcct
4441 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
4501 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
4561 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
4621 gttgaacaag tctggaaaga aatgcataag cttttgccat tctcaccgga ttcagtcgtc
4681 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
4741 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
4801 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
4861 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagta
4921 ctgacaataa aaagattctt gttttcaaga acttgtcatt tgtatagttt ttttatattg
4981 tagttgttct attttaatca aatgttagcg tgatttatat tttttttcgc ctcgacatca
5041 tctgcccaga tgcgaagtta agtgcgcaga aagtaatatc atgcgtcaat cgtatgtgaa
5101 tgctggtcgc tatactgctg tcgattcgat actaacgccg ccatccagtg tcgaaaacga
5161 gctcgaatta attcctgggg gaacaacttc acagaatgtt ttgtcatatt gtcgaagtgg
5221 tcacaaaaca agagaagttc cgccaattat aaaaagggaa cccgtatatt tcagcttcac
5281 ggatgatttc cagggtgaga gtactgtata tgggcttacg atagaaggcc ataaaaattt
5341 cttgcttggc aacaaaatag aagtgaaatc atgtcgaggc tgctgtgtgg gagaacagca
5401 taaaatatca caaaaaaaga atctaaaaca ctgtgttgct tgtcccagaa agggaatcaa
5461 gtatttttat aaagattgga gtggtaaaaa tcgagtatgt gctagatgct atggaagata
5521 caaattcagc ggtcatcact gtataaattg caagtatgta ccagaagcac gtgaagtgaa
5581 aaaggcaaaa gacaaaggcg aaaaattggg cattacgccc gaaggtttgc cagttaaagg
5641 accagagtgt ataaaatgtg gcggaatctt acagtggcct atgcggccgc tctagctaga
5701 actagtggat cgatccccaa ttcgccctat agtgagtcgt attacaattc actggccgtc
5761 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
5821 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
5881 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
5941 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
6001 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
6061 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
6121 aagctctaaa tcggggtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
6181 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
6241 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
6301 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
6361 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
6421 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
6481 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
6541 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
6601 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
6661 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
6721 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
6781 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
6841 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
6901 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
6961 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
7021 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
7081 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
7141 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
7201 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
7261 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
7321 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
7381 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
7441 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
7501 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
7561 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
7621 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
7681 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
7741 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
7801 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
7861 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
7921 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
7981 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
8041 ccgggttgga ctcaagacga tagttaccgg ataaggcaca gcggtcgggc tgaacggggg
8101 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
8161 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
8221 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
8281 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
8341 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
8401 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
8461 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
8521 agtcagtgag cgaggaagcg gaagagcgcc caat
//
