| 质粒名称: | pBHRSF184 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 89482 |
| 存储实验室: | Hideo Iwai |
|
载体骨架基本信息 |
|
| 载体名称: | pHYRSF53 |
| 载体出品公司: | -- |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | 4540 bp |
| 完整质粒大小: | 5245 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 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: | Asparaginyl endopeptidase from Oldenlandia affinis,OaAEP1 |
| 物种来源: | Synthetic |
| 插入大小: | 1353 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | T7 |
| 标签/融合蛋白: | Smt3（主干上的N终端） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | BamHI (未破坏) |
| 3' 克隆位点: | HindIII (未破坏) |
| 5' 测序引物 | T7 |
| 3' 测序引物 | T7term |
|
文献引用方法 |
|
| 材料和方法部分: | pBHRSF184 was a gift from Hideo Iwai (Addgene plasmid # 89482 ; http://n2t.net/addgene:89482 ; RRID:Addgene_89482) |
| 参考文献部分: | Segmental isotopic labeling of a single-domain globular protein without any refolding step by an asparaginyl endopeptidase. Mikula KM, Tascon I, Tommila JJ, Iwai H. FEBS Lett. 2017 Mar 30. doi: 10.1002/1873-3468.12640. 10.1002/1873-3468.12640 PubMed 28369872 |
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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 pBHRSF184 5245 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Express asparaginyl endopeptidase 1 from O. affinis (OaAEP1).
ACCESSION .
VERSION .
KEYWORDS pBHRSF184
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5245)
AUTHORS Mikula KM, Tascon I, Tommila JJ, Iwai H
TITLE Segmental isotopic labeling of a single-domain globular protein
without any refolding step by an asparaginyl endopeptidase.
JOURNAL FEBS Lett. 2017 Mar 30. doi: 10.1002/1873-3468.12640.
PUBMED 28369872
REFERENCE 2 (bases 1 to 5245)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..5245
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 1..20
/label=T7
/note="T7 promoter, forward primer"
promoter 1..19
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 20..44
/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 100..117
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 127..411
/codon_start=1
/gene="S. cerevisiae SMT3 (truncated)"
/product="cleavable ubiquitin-like protein tag"
/label=SUMO
/translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
LMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG"
CDS 1799..1843
/codon_start=1
/product="affinity and epitope tag derived from pancreatic
ribonuclease A"
/label=S-Tag
/translation="KETAAAKFERQHMDS"
primer_bind complement(1881..1899)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 1895..1942
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(2172..2987)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind 2895..2914
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
promoter complement(2988..3079)
/gene="bla"
/label=AmpR promoter
rep_origin complement(3095..3844)
/direction=LEFT
/label=RSF ori
/note="Plasmids containing the RSF 1030 origin of
replication can be propagated in E. coli cells that contain
additional plasmids with compatible origins."
primer_bind complement(3105..3122)
/label=L4440
/note="L4440 vector, forward primer"
primer_bind complement(3345..3364)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4040..5122)
/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 5084..5103
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(5123..5200)
/gene="lacI"
/label=lacI promoter
ORIGIN
1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
61 tgtttaactt taataaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
121 actagtgact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct
181 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag aaatcttctt taagatcaaa
241 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg
301 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat
361 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggatccgct
421 cgcgatggtg attatctgca tctgccgagc gaagtgagcc gtttctttcg cccgcaggaa
481 acaaacgatg atcatggtga agatagcgtg ggtacccgct gggcggtgct gattgcgggc
541 tctaaaggct atgcgaacta tcgccatcag gctggcgtgt gccatgcgta tcagattctg
601 aaacgcggcg gcctgaaaga tgaaaacatt gttgtgttca tgtatgatga tattgcgtat
661 aacgaaagca atccacgtcc gggcgtgatt attaacagcc cgcatggtag cgatgtttat
721 gctggcgtgc cgaaagatta tacaggcgaa gaagtgaacg cgaaaaactt tctggcggcg
781 attctgggca acaaaagcgc gattaccggc ggcagcggca aagtggtgga tagcggtccg
841 aacgaccata tttttattta ttataccgat catggcgctg cgggcgtgat tggcatgccg
901 agcaaaccgt atctgtatgc agatgaactg aacgatgcgc tgaaaaagaa acatgcgagc
961 ggtacatata aaagcctggt gttttatctg gaagcgtgcg aaagcggcag catgtttgaa
1021 ggtattttgc cggaagattt gaacatttat gcgctgacca gcaccaacac caccgaaagc
1081 agctggtgct attattgtcc ggcgcaggaa aacccgccac cgccggaata taacgtgtgc
1141 ctgggcgatc tgtttagcgt ggcgtggctg gaagatagcg atgtgcagaa ttcctggtat
1201 gaaaccctga accagcagta tcatcatgtg gataaacgca ttagccatgc gagccatgcg
1261 acccagtatg gcaacctgaa attgggcgaa gaaggcctgt ttgtgtatat gggcagcaac
1321 ccggcgaacg ataactatac ctctctggat ggcaacgcgc tgaccccgtc atcgattgtg
1381 gtgaaccagc gcgatgcgga tctgctgcat ctgtgggaaa aattccgcaa agcgccggaa
1441 ggcagcgcgc gcaaagaaga agcgcagacc cagattttta aagcgatgag ccatcgcgtg
1501 catattgata gcagcattaa actgattggc aaactgctgt ttggcattga aaaatgcacc
1561 gaaattctga acgcggttcg tccggcaggt caaccgctgg tggatgattg ggcgtgcctg
1621 cgtagcctgg tgggcacctt tgaaacccat tgcggcagcc tgagcgaata tggcatgcgc
1681 catacccgca ccattgcgaa catttgcaac gcgggcatta gcgaagaaca gatggcggaa
1741 gcggcgagcc aggcgtgcgc gagcattccg taagcttgcg gccgcactcg agtctggtaa
1801 agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta
1861 attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
1921 acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact
1981 gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc
2041 ctgccctgaa ccgacgacaa gctgacgacc gggtctccgc aagtggcact tttcggggaa
2101 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
2161 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
2221 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
2281 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
2341 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
2401 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
2461 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
2521 attcgtgatt gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg acaattacaa
2581 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
2641 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
2701 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
2761 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
2821 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
2881 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
2941 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcatact cttccttttt
3001 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
3061 atttagaaaa ataaacaaat aggcatgcag cgctcttccg cttcctcgct cactgactcg
3121 ctacgctcgg tcgttcgact gcggcgagcg gtgtcagctc actcaaaagc ggtaatacgg
3181 ttatccacag aatcagggga taaagccgga aagaacatgt gagcaaaaag caaagcaccg
3241 gaagaagcca acgccgcagg cgtttttcca taggctccgc ccccctgacg agcatcacaa
3301 aaatcgacgc tcaagccaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
3361 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
3421 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gttggtatct
3481 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
3541 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
3601 atcgccactg gcagcagcca ttggtaactg atttagagga ctttgtcttg aagttatgca
3661 cctgttaagg ctaaactgaa agaacagatt ttggtgagtg cggtcctcca acccacttac
3721 cttggttcaa agagttggta gctcagcgaa ccttgagaaa accaccgttg gtagcggtgg
3781 tttttcttta tttatgagat gatgaatcaa tcggtctatc aagtcaacga acagctattc
3841 cgttactcta gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca
3901 tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga aggagctgac
3961 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
4021 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
4081 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
4141 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
4201 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
4261 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
4321 tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
4381 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
4441 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
4501 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
4561 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
4621 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
4681 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
4741 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
4801 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
4861 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
4921 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
4981 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
5041 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
5101 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
5161 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
5221 ttatgcgact cctgcattag gaaat
//
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