| 质粒名称: | Flag-TurboID |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 124646 |
| 存储实验室: | FENG-QIAN Li, Ken-Ichi Takemaru |
|
载体骨架基本信息 |
|
| 载体名称: | pCD-betaG-FLAG |
| 完整质粒大小: | 5096 bp |
| 载体类型: | 哺乳细胞表达 |
| 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: | TurboID |
| 启动子: | CMV |
| 标签/融合蛋白: | Flag (N terminal on backbone) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BglII (destroyed during cloning) |
| 3’克隆位点: | BglII (未破坏) |
| 5’测序引物 | GCGTGCCTAATGGGAGGTCT |
|
文献引用方法 |
|
| 材料和方法部分: | Flag-TurboID was a gift from FENG-QIAN Li & Ken-Ichi Takemaru (Addgene plasmid # 124646 ; http://n2t.net/addgene:124646 ; RRID:Addgene_124646) |
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 Flag-TurboID 5096 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses Flag-TurboID fusion for proximity labeling.
ACCESSION .
VERSION .
KEYWORDS Flag-TurboID
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5096)
TITLE Expresses Flag-TurboID fusion for proximity labeling
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 5096)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5096
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..978
/label=CMV IE94 promoter
/note="enhancer/promoter region of simian cytomegalovirus
major immediate early transcription unit IE94"
promoter 1013..1031
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 1013..1030
/label=SP6
/note="SP6 promoter, forward primer"
CDS 1069..1092
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 1099..2055
/codon_start=1
/product="mutant of the E. coli biotin ligase BioID
engineered for improved catalytic efficiency (Branon et
al., 2018)"
/label=TurboID
/translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
promoter complement(2104..2121)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
polyA_signal 2126..2260
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2150..2169)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 2204..2223
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(2373..2393)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(2373..2391)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(2412..2428)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2412..2428)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(2425..2447)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 2436..2452
/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)."
promoter complement(2460..2490)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2505..2526
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(2643..2660)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(2814..3402)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(2894..2913)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(3573..4433)
/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 4196..4215
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(4434..4538)
/gene="bla"
/label=AmpR promoter
rep_origin complement(4564..5019)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4701..4722)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 4913..4932
/label=F1ori-R
/note="F1 origin, reverse primer"
ORIGIN
1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt
1021 gacactatag aatacaagct acttgttctt tttgcaggat ccaccatgga ttacaaggat
1081 gacgacgata agatccgcaa agacaatact gtgcctctga agctgatcgc tctcctggct
1141 aatggcgagt tccatagtgg cgaacagctg ggagaaaccc tgggcatgtc cagggccgct
1201 atcaacaagc acattcagac tctgcgcgac tggggcgtgg acgtgttcac cgtgcccgga
1261 aagggctact ctctgcccga gcctatcccg ctgctgaacg ctaaacagat tctgggacag
1321 ctggacggcg ggagcgtggc agtcctgcct gtggtcgact ccaccaatca gtacctgctg
1381 gatcgaatcg gcgagctgaa gagtggggat gcttgcattg cagaatatca gcaggcaggg
1441 agaggaagca gagggaggaa atggttctct ccttttggag ctaacctgta cctgagtatg
1501 ttttggcgcc tgaagcgggg accagcagca atcggcctgg gcccggtcat cggaattgtc
1561 atggcagaag cgctgcgaaa gctgggagca gacaaggtgc gagtcaaatg gcccaatgac
1621 ctgtatctgc aggatagaaa gctggcaggc atcctggtgg agctggccgg aataacaggc
1681 gatgctgcac agatcgtcat tggcgccggg attaacgtgg ctatgaggcg cgtggaggaa
1741 agcgtggtca atcagggctg gatcacactg caggaagcag ggattaacct ggacaggaat
1801 actctggccg ctacgctgat ccgagagctg cgggcagccc tggaactgtt cgagcaggaa
1861 ggcctggctc catatctgcc acggtgggag aagctggata acttcatcaa tagacccgtg
1921 aagctgatca ttggggacaa agagattttc gggattagcc gggggattga taaacaggga
1981 gccctgctgc tggaacagga cggagttatc aaaccctgga tgggcggaga aatcagtctg
2041 cggtctgccg aaaagctgca gggatctcag atctcgaatt caaggcctct cgagcctcta
2101 gaactatagt gagtcgtatt acgtagatcc agacatgata agatacattg atgagtttgg
2161 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
2221 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
2281 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taattcgcgg ccgcggcgcc
2341 aatgcattgg gcccggtacc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
2401 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
2461 aacatacgag ccgggagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
2521 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
2581 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
2641 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
2701 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
2761 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
2821 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
2881 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
2941 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
3001 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
3061 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
3121 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
3181 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
3241 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
3301 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
3361 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
3421 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
3481 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
3541 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
3601 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
3661 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
3721 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
3781 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
3841 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
3901 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
3961 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
4021 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
4081 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
4141 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
4201 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
4261 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
4321 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
4381 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
4441 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
4501 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
4561 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
4621 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
4681 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
4741 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
4801 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
4861 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
4921 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
4981 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
5041 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag tcgacc
//
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