| 质粒名称: | pGEX6p-1-FRB-PafA |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 113404 |
| 存储实验室: | Min Zhuang |
|
载体骨架基本信息 |
|
| 载体名称: | pGEX6P-1 |
| 完整质粒大小: | 6759 bp |
| 载体类型: | 细菌表达 |
| 载体抗性: | 氨苄霉素 |
| 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: | FRB-PafA |
| 物种来源: | 未知 |
| 插入大小: | 1785bp |
| 启动子: | TAC |
| 标签/融合蛋白: | GST (N terminal on backbone) |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | self-designed |
|
文献引用方法 |
|
| 材料和方法部分: | pGEX6p-1-FRB-PafA was a gift from Min Zhuang (Addgene plasmid # 113404 ; http://n2t.net/addgene:113404 ; RRID:Addgene_113404) |
| 参考文献部分: | A proximity-tagging system to identify membrane protein-protein interactions. Liu Q, Zheng J, Sun W, Huo Y, Zhang L, Hao P, Wang H, Zhuang M. Nat Methods. 2018 Aug 13. pii: 10.1038/s41592-018-0100-5. doi: 10.1038/s41592-018-0100-5. 10.1038/s41592-018-0100-5 PubMed 30104635 |
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 pGEX6p-1-FRB-PafA 6759 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses FRB-PafA in bacteria.
ACCESSION .
VERSION .
KEYWORDS pGEX6p-1-FRB-PafA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6759)
AUTHORS Liu Q, Zheng J, Sun W, Huo Y, Zhang L, Hao P, Wang H, Zhuang M
TITLE A proximity-tagging system to identify membrane protein-protein
interactions.
JOURNAL Nat Methods. 2018 Aug 13. pii: 10.1038/s41592-018-0100-5. doi:
10.1038/s41592-018-0100-5.
PUBMED 30104635
REFERENCE 2 (bases 1 to 6759)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6759
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 1..654
/codon_start=1
/product="glutathione S-transferase from Schistosoma
japonicum"
/label=GST
/translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
primer_bind 612..634
/label=pGEX 5'
/note="pGEX vectors, Glutathione-S-transferase, forward
primer"
CDS 661..684
/codon_start=1
/product="recognition and cleavage site for human
rhinovirus 3C and PreScission proteases"
/label=HRV 3C site
/translation="LEVLFQGP"
CDS 709..978
/codon_start=1
/product="FKBP-rapamycin binding domain of human FRAP"
/label=FRB
/translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
primer_bind complement(2552..2574)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(2719..2737)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2805..2909
/gene="bla"
/label=AmpR promoter
CDS 2910..3770
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(3128..3147)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 3941..4529
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4430..4449
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4683..4700
/label=L4440
/note="L4440 vector, forward primer"
promoter 4773..4850
/gene="lacI"
/label=lacI promoter
CDS 4851..5933
/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(4870..4889)
/label=LacI-R
/note="LacI, reverse primer"
promoter 5982..6012
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6020..6036
/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 6044..6060
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 6044..6060
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
CDS 6056..6583
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/label=lacZ-alpha
/translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
QPSEAVVWLCRS"
primer_bind complement(6076..6093)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(6076..6092)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6085..6107)
/label=M13/pUC Forward
/note="In lacZ gene"
promoter 6685..6713
/label=tac promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 6721..6737
/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 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
61 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
121 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat
181 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
241 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg
301 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt
361 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
421 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat
481 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
541 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca
601 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat
661 ctggaagttc tgttccaggg gcccctggga tccccgattc tgtggcatga gatgtggcac
721 gagggactcg aagaggccag caggctgtac tttggcgaga ggaacgtcaa gggcatgttc
781 gaagtgctgg agcccctcca tgcgatgatg gaaaggggcc cacagaccct gaaggagacc
841 agcttcaacc aggcttacgg cagggacctg atggaggcac aggaatggtg caggaagtac
901 atgaagagcg gcaacgtgaa agacctgacc caggcgtggg acctctacta ccacgtgttc
961 aggaggatca gcaagcagtc cggtggcggc agcggtgggg gcagtggagg cggaagcgga
1021 gggggctctg gcatgagtac cgtggaatcc gcattgaccc gcaggatcat gggcattgaa
1081 acggagtatg gcctcacctt tgttgatggt gattccaaaa agcttcgccc agatgagata
1141 gctcgaagga tgtttcgtcc catcgtggag aaatattcca gctctaatat cttcataccc
1201 aatggttccc ggttgtatct tgatgtgggt tcccacccgg agtacgccac cgccgagtgt
1261 gataatttga cccagctgat caattttgaa aaagctggcg atgttattgc agatcgcatg
1321 gctgtagatg ccgaagagtc gctggcgaaa gaagacattg ctgggcaggt gtacctgttt
1381 aaaaacaatg tcgattccgt gggcaattct tatggctgcc acgaaaacta ccttgtgggt
1441 cgctccatgc cgttgaaggc gttgggtaaa aggctgatgc cgtttctgat tacccgccag
1501 ctcatctgcg gcgccggcag gatccatcac cccaatcctt tggataaagg cgaatccttc
1561 cccttgggct actgcatatc ccagcgctct gaccacgtgt gggagggcgt atcaagtgcc
1621 accactagat cacgccccat tatcaacacc cgtgatgagc cacatgcgga ttcccattct
1681 taccgcaggc tgcacgtgat tgtgggtgat gccaacatgg cagagcccag catcgcgttg
1741 aaggtcggct ccacgttgct ggttctggaa atgattgagg cagatttcgg tttgcccagc
1801 ttagagcttg ccaatgatat tgcctcaatt agggaaatct cccgcgatgc aacaggatcc
1861 acactgttgt ccctgaaaga tggcaccacc atgactgcct tgcagatcca gcaggtggtc
1921 tttgagcatg cctcgaagtg gttggagcag cgccccgaac cagaattttc tggcacctcc
1981 aacacagaga tggcccgcgt gctggatctg tggggtcgca tgttgaaagc gattgagtcc
2041 ggtgatttca gcgaagtgga tacagaaatt gactgggtga tcaaaaagaa gctcattgat
2101 cgtttcattc agcgcggcaa ccttgggttg gatgatccaa aacttgccca agtggacttg
2161 acttatcacg atattaggcc aggtagaggc ctatttagcg tgctgcaaag ccgcggcatg
2221 atcaaacggt ggactactga tgaggcgatt ttagctgcgg tggataccgc tcctgataca
2281 acacgtgctc atttgcgcgg gcgaatcctt aaagcggcgg atactctggg agtacctgtg
2341 actgtcgatt ggatgcgtca caaggtcaac cgaccggagc cacaatcggt ggaattgggg
2401 gatccttttt ccgcagtgaa ttccgaggtt gatcagttga ttgagtatat gaccgttcat
2461 gcggagagct atcgcagtta agtcgactcg agcggccgca tcgtgactga ctgacgatct
2521 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
2581 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
2641 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
2701 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
2761 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
2821 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
2881 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
2941 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
3001 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
3061 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
3121 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
3181 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
3241 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
3301 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
3361 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
3421 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
3481 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
3541 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
3601 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
3661 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
3721 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
3781 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
3841 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
3901 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
3961 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
4021 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
4081 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
4141 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
4201 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
4261 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
4321 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
4381 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
4441 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
4501 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
4561 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
4621 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
4681 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
4741 atctgtgcgg tatttcacac cgcataaatt ccgacaccat cgaatggcgc aaaacctttc
4801 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
4861 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
4921 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
4981 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
5041 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
5101 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
5161 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
5221 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
5281 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
5341 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
5401 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
5461 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
5521 ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg
5581 ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg
5641 ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc
5701 cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct
5761 tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg
5821 tgaaaagaaa aaccacgcta gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
5881 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
5941 gcaattaatg taagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
6001 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
6061 catgattacg gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
6121 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
6181 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc
6241 ctggtttccg gcaccagaag cggtgccgga aagctggctg gagtgcgatc ttcctgaggc
6301 cgatactgtc gtcgtcccct caaactggca gatgcacggt tacgatgcgc ccatctacac
6361 caacgtgacc tatcccatta cggtcaatcc gccgtttgtt cccacggaga atccgacggg
6421 ttgttactcg ctcacattta atgttgatga aagctggcta caggaaggcc agacgcgaat
6481 tatttttgat ggcgttggaa ttagcttatc gactgcacgg tgcaccaatg cttctggcgt
6541 caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt
6601 gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc
6661 tggcaaatat tctgaaatga gctgttgaca attaatcatc ggctcgtata atgtgtggaa
6721 ttgtgagcgg ataacaattt cacacaggaa acagtattc
//
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