| 质粒名称: | pRSFDuet-1_VNp-mNeongreen-Erythropoietin |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 182423 |
| 存储实验室: | Dan Mulvihill |
|
载体骨架基本信息 |
|
| 载体名称: | pRSFDUET-1 |
| 载体出品公司: | Novagen |
| 空载体大小: | 8700 bp |
| 完整质粒大小: | 9790 bp |
| 载体类型: | 细菌 |
| 载体抗性: | Kanamycin |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5alpha |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | VNp-mNeongreen-eythropoietin |
| 物种来源: | H. sapiens (human) |
| 基因编号: | UniProtKB: P01588 |
| 启动子: | TRE3G |
| 融合/标签： | Vesicle Nucleating peptide (VNp) & mNeongreen (N terminal on insert) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | Unknown (unknown if destroyed) |
| 3’克隆位点: | Unknown (unknown if destroyed) |
|
文献引用方法 |
|
| 材料和方法部分: |
pRSFDuet-1_VNp-mNeongreen-Erythropoietin was a gift from Dan Mulvihill (Addgene plasmid # 182423 ; http://n2t.net/addgene:182423 ; RRID:Addgene_182423)
|
| 参考文献部分: |
High-yield vesicle-packaged recombinant protein production from E. coli. Eastwood TA, Baker K, Streather BR, Allen N, Wang L, Botchway SW, Brown IR, Hiscock JR, Lennon C, Mulvihill DP. Cell Rep Methods. 2023 Feb 2;3(2):100396. doi: 10.1016/j.crmeth.2023.100396. eCollection 2023 Feb 27. 10.1016/j.crmeth.2023.100396 PubMed 36936078
|
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 pRSFDuet-1_VNp-mNeongreen-Erythropoietin 5164 bp ds-DNA circular SYN 02-MAY-2022
DEFINITION Bacterial expression of Vesicle Nucleating
peptide-mNeongreen-Erythropoietin fusion.
ACCESSION .
VERSION .
KEYWORDS pRSFDuet-1_VNp-mNeongreen-Erythropoietin
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5164)
AUTHORS Eastwood TA, Baker K, Allen N, Wang L, Botchway SW, Brown IR,
Hiscock JR, Lennon C, Mulvihill DP
TITLE Exceptional yield vesicular recombinant protein production from E.
coli.
JOURNAL unpublished
REFERENCE 2 (bases 1 to 5164)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 2, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5164
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 3..27
/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 83..100
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
promoter 214..232
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 233..257
/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 432..1139
/codon_start=1
/product="bright monomeric yellow-green fluorescent protein
derived from LanYFP (Shaner et al., 2013)"
/label=mNeonGreen
/translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
KAFTDVMGMDELYK"
CDS 1668..1685
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 1701..1745
/codon_start=1
/product="affinity and epitope tag derived from pancreatic
ribonuclease A"
/label=S-Tag
/translation="KETAAAKFERQHMDS"
primer_bind complement(1783..1801)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 1797..1844
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(2074..2889)
/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 2797..2816
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
promoter complement(2890..2981)
/gene="bla"
/label=AmpR promoter
rep_origin complement(2997..3746)
/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(3007..3024)
/label=L4440
/note="L4440 vector, forward primer"
primer_bind complement(3247..3266)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(3942..5024)
/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 4986..5005
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(5025..5102)
/gene="lacI"
/label=lacI promoter
promoter 5148..2
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
301 tggatgtatt catgaaagga ctttcaaagg ccaaggaggg agttgtggca gctgctgaga
361 aaaccaaaca gggtgtggca gaagcagcag gaaagacaaa agagggtgtt ctcagatctg
421 gaggaagcgg aatggtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
481 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
541 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
601 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
661 accctgacgg gatgtcgcct ttccaggccg cgatggtaga tggctccgga taccaagtcc
721 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
781 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
841 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
901 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
961 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
1021 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
1081 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagg
1141 ctagcagatc tggaggaagc ggagcaccgc ctcgtctgat ttgtgatagc cgtgttctgg
1201 aacgttatct gctggaagca aaagaagcag aaaacattac caccggttgt gcagaacatt
1261 gtagcctgaa tgaaaatatc accgttccgg ataccaaagt gaacttttat gcatggaaac
1321 gtatggaagt tggtcagcag gcagttgaag tttggcaagg tctggcactg ctgagcgaag
1381 cagttctgcg tggtcaggcc ctgctggtta atagcagcca gccgtgggaa ccgctgcaac
1441 tgcatgttga taaagcagtt agcggtctgc gtagcctgac aaccctgctg cgtgcactgg
1501 gtgcacagaa agaagcaatt agccctccgg atgcagcaag cgcagcaccg ctgcgtacca
1561 ttaccgcaga tacctttcgt aaactgtttc gcgtgtatag caatttcctg cgtggtaaac
1621 tgaaactgta taccggtgaa gcatgtcgta ccggtgatcg tggtacccat catcatcatc
1681 atcattaact cgagtctggt aaagaaaccg ctgctgcgaa atttgaacgc cagcacatgg
1741 actcgtctac tagcgcagct taattaacct aggctgctgc caccgctgag caataactag
1801 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa cctcaggcat
1861 ttgagaagca cacggtcaca ctgcttccgg tagtcaataa accggtaaac cagcaataga
1921 cataagcggc tatttaacga ccctgccctg aaccgacgac aagctgacga ccgggtctcc
1981 gcaagtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
2041 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
2101 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
2161 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
2221 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
2281 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
2341 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
2401 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcggtc
2461 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
2521 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
2581 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
2641 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
2701 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
2761 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
2821 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
2881 atggctcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
2941 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggcatgc agcgctcttc
3001 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggtgtcagc
3061 tcactcaaaa gcggtaatac ggttatccac agaatcaggg gataaagccg gaaagaacat
3121 gtgagcaaaa agcaaagcac cggaagaagc caacgccgca ggcgtttttc cataggctcc
3181 gcccccctga cgagcatcac aaaaatcgac gctcaagcca gaggtggcga aacccgacag
3241 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
3301 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
3361 atagctcacg ctgttggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
3421 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
3481 ccaacccggt aagacacgac ttatcgccac tggcagcagc cattggtaac tgatttagag
3541 gactttgtct tgaagttatg cacctgttaa ggctaaactg aaagaacaga ttttggtgag
3601 tgcggtcctc caacccactt accttggttc aaagagttgg tagctcagcg aaccttgaga
3661 aaaccaccgt tggtagcggt ggtttttctt tatttatgag atgatgaatc aatcggtcta
3721 tcaagtcaac gaacagctat tccgttactc tagatttcag tgcaatttat ctcttcaaat
3781 gtagcacctg aagtcagccc catacgatat aagttgtaat tctcatgtta gtcatgcccc
3841 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
3901 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
3961 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
4021 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
4081 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
4141 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
4201 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg
4261 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
4321 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
4381 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
4441 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
4501 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
4561 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
4621 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
4681 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
4741 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
4801 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
4861 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
4921 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
4981 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
5041 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
5101 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaaattaa tacgactcac
5161 tata
//
