| 质粒名称: | pTXB1-APEX2-F |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 160083 |
| 存储实验室: | Pier Pandolfi |
|
载体骨架基本信息 |
|
| 载体名称: | pTXB1 |
| 载体出品公司: | New England Biolabs |
| 空载体大小: | 6706 bp |
| 完整质粒大小: | 7428 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: | APEX2 peroxidase |
| 物种来源: | Synthetic |
| 插入大小: | 2175 bp |
| 标签/融合蛋白: |
FLAG (C terminal on insert)
Mxe intein - Chitin-binding domain (C terminal on insert)
|
| 克隆方法: | 未知 |
| 5’克隆位点: | AgeI(未破坏) |
| 5’测序引物 | T7 promoter primer |
| 3’测序引物 | GATTGCCATGCCGGTCAAGG |
|
文献引用方法 |
|
| 材料和方法部分: | pTXB1-APEX2-F was a gift from Pier Pandolfi (Addgene plasmid # 160083 ; http://n2t.net/addgene:160083 ; RRID:Addgene_160083) |
| 参考文献部分: | Dual DNA and protein tagging of open chromatin unveils dynamics of epigenomic landscapes in leukemia. Lee JD, Paulo JA, Posey RR, Mugoni V, Kong NR, Cheloni G, Lee YR, Slack FJ, Tenen DG, Clohessy JG, Gygi SP, Pandolfi PP. Nat Methods. 2021 Mar;18(3):293-302. doi: 10.1038/s41592-021-01077-8. Epub 2021 Mar 1. 10.1038/s41592-021-01077-8 PubMed 33649590 |
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 pTXB1-APEX2-F 7428 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Bacterial expression of C-terminally FLAG-tagged APEX2 peroxidase.
ACCESSION .
VERSION .
KEYWORDS pTXB1-APEX2-F
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7428)
TITLE Dual DNA/protein tagging of open chromatin unveils dynamics of
epigenomic landscapes in leukemia
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 7428)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7428
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 126..148
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(142..333)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(890..1972)
/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 1934..1953
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(1973..2050)
/gene="lacI"
/label=lacI promoter
terminator 2203..2289
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 2386..2472
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 2569..2655
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 2752..2838
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 2935..3021
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
primer_bind 3156..3175
/label=T7
/note="T7 promoter, forward primer"
promoter 3156..3174
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 3175..3199
/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)."
CDS 3243..3992
/codon_start=1
/product="soybean ascorbate peroxidase, engineered to serve
as a monomeric tag for electron microscopy and live-cell
proteomics (Lam et al., 2015)"
/label=APEX2
/note="more active than the original APEX due to the A134P
mutation"
/translation="MGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAG
TFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVA
VEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGH
TIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVD
KYAADEDAFFADYAEAHQKLSELGFADA"
CDS 3993..4016
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 4017..4610
/codon_start=1
/gene="Mycobacterium xenopi gyrA"
/product="modified GyrA intein (Southworth et al., 1999)"
/label=Mxe GyrA intein
/note="N-terminal cleavage of this intein can be induced by
thiols."
/translation="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV
LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
CDS 4641..4796
/codon_start=1
/gene="Bacillus circulans chiA"
/product="chitin binding domain from chitinase A1 (Watanabe
et al., 1994)"
/label=CBD
/translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
PALWQLQ"
primer_bind complement(4861..4879)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 4875..4922
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter 4982..5086
/gene="bla"
/label=AmpR promoter
CDS 5087..5947
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(5305..5324)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin complement(5989..6502)
/direction=LEFT
/label=M13 ori
/note="M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(6184..6205)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 6396..6415
/label=F1ori-R
/note="F1 origin, reverse primer"
rep_origin 6613..7201
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7102..7121
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 7355..7372
/label=L4440
/note="L4440 vector, forward primer"
primer_bind complement(7394..7413)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
61 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
121 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
181 gctgcggtaa agctcatcag cgtggtcgtg cagcgattca cagatgtctg cctgttcatc
241 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
301 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
361 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
421 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
481 gcgggaccag agaaaaatca ctcagggtca atgccagccg aacgccagca agacgtagcc
541 cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
601 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
661 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
721 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
781 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
841 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
901 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
961 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
1021 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
1081 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
1141 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
1201 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
1261 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
1321 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
1381 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
1441 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
1501 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
1561 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
1621 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
1681 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
1741 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
1801 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
1861 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
1921 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
1981 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
2041 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
2101 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg ccggcatgcc
2161 gccctttcgt cttcaagaat taattcccaa ttccccaggc atcaaataaa acgaaaggct
2221 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
2281 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
2341 gcaggacgcc cgccataaac tgccaggaat taattcccca ggcatcaaat aaaacgaaag
2401 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
2461 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
2521 cgggcaggac gcccgccata aactgccagg aattaattcc ccaggcatca aataaaacga
2581 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
2641 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
2701 tggcgggcag gacgcccgcc ataaactgcc aggaattaat tccccaggca tcaaataaaa
2761 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
2821 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
2881 gggtggcggg caggacgccc gccataaact gccaggaatt aattccccag gcatcaaata
2941 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
3001 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
3061 ggagggtggc gggcaggacg cccgccataa actgccagga attggggatc ggaattaatt
3121 cccggtttaa accggggatc tcgatcccgc gaaattaata cgactcacta taggggaatt
3181 gtgagcggat aacaattccc ctctagaaat aattttgtta actttaagaa ggagatatac
3241 atatgggaaa gtcttaccca actgtgagtg ctgattacca ggacgccgtt gagaaggcga
3301 agaagaagct cagaggcttc atcgctgaga agagatgcgc tcctctaatg ctccgtttgg
3361 cattccactc tgctggaacc tttgacaagg gcacgaagac cggtggaccc ttcggaacca
3421 tcaagcaccc tgccgaactg gctcacagcg ctaacaacgg tcttgacatc gctgttaggc
3481 ttttggagcc actcaaggcg gagttcccta ttttgagcta cgccgatttc taccagttgg
3541 ctggcgttgt tgccgttgag gtcacgggtg gacctaaggt tccattccac cctggaagag
3601 aggacaagcc tgagccacca ccagagggtc gcttgcccga tcccactaag ggttctgacc
3661 atttgagaga tgtgtttggc aaagctatgg ggcttactga ccaagatatc gttgctctat
3721 ctgggggtca cactattgga gctgcacaca aggagcgttc tggatttgag ggtccctgga
3781 cctctaatcc tcttattttc gacaactcat acttcacgga gttgttgagt ggtgagaagg
3841 aaggtctcct tcagctacct tctgacaagg ctcttttgtc tgaccctgta ttccgccctc
3901 tcgttgacaa atatgcagcg gacgaagatg ccttctttgc tgattacgct gaggctcacc
3961 aaaagctttc cgagcttggg tttgctgatg ccgattacaa ggatgacgac gataagtgca
4021 tcacgggaga tgcactagtt gccctacccg agggcgagtc ggtacgcatc gccgacatcg
4081 tgccgggtgc gcggcccaac agtgacaacg ccatcgacct gaaagtcctt gaccggcatg
4141 gcaatcccgt gctcgccgac cggctgttcc actccggcga gcatccggtg tacacggtgc
4201 gtacggtcga aggtctgcgt gtgacgggca ccgcgaacca cccgttgttg tgtttggtcg
4261 acgtcgccgg ggtgccgacc ctgctgtgga agctgatcga cgaaatcaag ccgggcgatt
4321 acgcggtgat tcaacgcagc gcattcagcg tcgactgtgc aggttttgcc cgcgggaaac
4381 ccgaatttgc gcccacaacc tacacagtcg gcgtccctgg actggtgcgt ttcttggaag
4441 cacaccaccg agacccggac gcccaagcta tcgccgacga gctgaccgac gggcggttct
4501 actacgcgaa agtcgccagt gtcaccgacg ccggcgtgca gccggtgtat agccttcgtg
4561 tcgacacggc agaccacgcg tttatcacga acgggttcgt cagccacgct actggcctca
4621 ccggtctgaa ctcaggcctc acgacaaatc ctggtgtatc cgcttggcag gtcaacacag
4681 cttatactgc gggacaattg gtcacatata acggcaagac gtataaatgt ttgcagcccc
4741 acacctcctt ggcaggatgg gaaccatcca acgttcctgc cttgtggcag cttcaatgac
4801 tgcaggaagg ggatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
4861 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
4921 tgctgaaagg aggaactata tccggataac tacgtcaggt ggcacttttc ggggaaatgt
4981 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
5041 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
5101 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
5161 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
5221 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgtttccc
5281 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
5341 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
5401 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
5461 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
5521 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
5581 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
5641 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
5701 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
5761 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
5821 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
5881 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
5941 ttggtaactg tcagaccaag tttactcata tatactttag attgatttac cccggttgat
6001 aatcagaaaa gccccaaaaa caggaagatt gtataagcaa atatttaaat tgtaaacgtt
6061 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
6121 gccgaaatcg gcaaaatccc ttataaatca aaagaatagc ccgagatagg gttgagtgtt
6181 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
6241 aaaaccgtct atcagggcga tggcccacta cgtgaaccat cacccaaatc aagttttttg
6301 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
6361 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
6421 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
6481 aatgcgccgc tatagggcgc gtaaaaggat ctaggtgaag atcctttttg ataatctcat
6541 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
6601 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
6661 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
6721 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
6781 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
6841 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
6901 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
6961 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
7021 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
7081 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
7141 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
7201 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
7261 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
7321 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagctat
7381 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagt
//
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