| 质粒名称: | pRSFDuet-1_VNp-mNeongreen_OmpA-mCherry |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 182390 |
| 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 |
| 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1: | VNp-mNeongreen-His |
| 物种来源: | Branchiostoma lanceolatum |
| GenBank编号 | UniProtKB: A0A1S4NYF2 |
| 启动子: | T7 |
| 标签/融合蛋白: | Vesicle Nucleating peptide (VNp) (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) |
| 5’测序引物 | n/a |
| 插入基因名称2: | OmpA-mCherry |
| 标签/融合蛋白: | T7 |
| 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) |
| 5’测序引物 | n/a |
|
文献引用方法 |
|
| 材料和方法部分: | pRSFDuet-1_VNp-mNeongreen_OmpA-mCherry was a gift from Dan Mulvihill (Addgene plasmid # 182390 ; http://n2t.net/addgene:182390 ; RRID:Addgene_182390) |
| 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_OmpA-mCherry 7818 bp ds-DNA circular SYN 22-JUN-2022
DEFINITION Bacterial expression of Vesicle Nucleating peptide-mNeongreen and
ompA-mCherry fusions.
ACCESSION .
VERSION .
KEYWORDS pRSFDuet-1_VNp-mNeongreen_OmpA-mCherry
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7818)
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JOURNAL unpublished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 7818)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jun 22, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7818
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 182..892
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
primer_bind complement(327..345)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 595..614
/label=mCherry-F
/note="mCherry, forward primer"
promoter 1001..1019
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 1020..1044
/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 1219..1926
/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 2269..2313
/codon_start=1
/product="affinity and epitope tag derived from pancreatic
ribonuclease A"
/label=S-Tag
/translation="KETAAAKFERQHMDS"
primer_bind complement(2351..2369)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 2365..2412
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 2449..2904
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2536..2555)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2746..2767
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(3019..3879)
/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 3642..3661
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(3880..3972)
/gene="bla"
/label=AmpR promoter
rep_origin 4053..4641
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4542..4561
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4795..4812
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 4827..4966
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(4892..4911)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5052..5074
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(5068..5259)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(5831..6913)
/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 6875..6894
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(6914..6991)
/gene="lacI"
/label=lacI promoter
primer_bind 7197..7216
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
promoter 7304..7322
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 7323..7347
/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)."
RBS 7362..7384
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
sig_peptide 7391..7453
/product="signal peptide from the E. coli outer membrane
protein OmpA"
/label=OmpA signal peptide
ORIGIN
1 cttcgctggc ggtgttgagt acgcgatcac tcctgaaatc gctacccgtc tggaatacca
61 gtggaccaac aacatcggtg acgcacacac catcggcact cgtccggaca acggcatgct
121 gagcctgggt gtttcctacc gtttcggtca gggcgaagga tccggtggtg gtggttcggg
181 tatggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
241 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
301 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
361 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
421 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
481 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
541 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
601 aatgcagaag aagactatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
661 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
721 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
781 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
841 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt aaagatccga
901 attcgagctc ggcgcgcctg caggtcgaca agcttgcggc cgcataatgc ttaagtcgaa
961 cagaaagtaa tcgtattgta cacggccgca taatcgaaat taatacgact cactataggg
1021 gaattgtgag cggataacaa ttccccatct tagtatatta gttaagtata agaaggagat
1081 atacatatgg atgtattcat gaaaggactt tcaaaggcca aggagggagt tgtggcagct
1141 gctgagaaaa ccaaacaggg tgtggcagaa gcagcaggaa agacaaaaga gggtgttctc
1201 agatctggag gaagcggaat ggtgagcaag ggcgaggagg ataacatggc ctctctccca
1261 gcgacacatg agttacacat ctttggctcc atcaacggtg tggactttga catggtgggt
1321 cagggcaccg gcaatccaaa tgatggttat gaggagttaa acctgaagtc caccaagggt
1381 gacctccagt tctccccctg gattctggtc cctcatatcg ggtatggctt ccatcagtac
1441 ctgccctacc ctgacgggat gtcgcctttc caggccgcga tggtagatgg ctccggatac
1501 caagtccatc gcacaatgca gtttgaagat ggtgcctccc ttactgttaa ctaccgctac
1561 acctacgagg gaagccacat caaaggagag gcccaggtga aggggactgg tttccctgct
1621 gacggtcctg tgatgaccaa ctcgctgacc gctgcggact ggtgcaggtc gaagaagact
1681 taccccaacg acaaaaccat catcagtacc tttaagtgga gttacaccac tggaaatggc
1741 aagcgctacc ggagcactgc gcggaccacc tacacctttg ccaagccaat ggcggctaac
1801 tatctgaaga accagccgat gtacgtgttc cgtaagacgg agctcaagca ctccaagacc
1861 gagctcaact tcaaggagtg gcaaaaggcc tttaccgatg tgatgggcat ggacgagctg
1921 tacaaggcta gcggaggaag cggaatgatg ttcttaccaa ccgattattg ttgcaggtta
1981 agcgatcaag agtatatgga gcttgtgttt gagaatggcc agattcttgc aaagggccaa
2041 agatccaacg tttctctgca taatcaacgt accaaatcga tcatggattt gtatgaggca
2101 gagtataacg aggatttcat gaagagtatc attcatggtg gtggtggtgc catcacaaat
2161 ctcggggaca cgcaggttgt tccacaaagt catgttgctg ctgcccatga aacaaacatg
2221 ttggaaagca ataaacatgt tgactaaact agttaactcg agtctggtaa agaaaccgct
2281 gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag
2341 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
2401 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
2461 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
2521 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
2581 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
2641 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
2701 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
2761 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
2821 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
2881 acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat
2941 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
3001 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
3061 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
3121 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
3181 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
3241 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
3301 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
3361 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
3421 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
3481 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
3541 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
3601 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
3661 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
3721 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
3781 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
3841 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg
3901 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
3961 aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
4021 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
4081 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
4141 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
4201 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
4261 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
4321 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
4381 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
4441 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
4501 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
4561 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
4621 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
4681 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
4741 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
4801 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
4861 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
4921 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
4981 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
5041 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
5101 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
5161 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
5221 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
5281 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
5341 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
5401 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
5461 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
5521 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
5581 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
5641 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
5701 caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
5761 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
5821 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
5881 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
5941 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
6001 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
6061 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
6121 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
6181 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
6241 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
6301 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
6361 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
6421 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
6481 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
6541 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
6601 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
6661 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
6721 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
6781 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
6841 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
6901 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
6961 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
7021 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
7081 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
7141 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
7201 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
7261 cgtccggcgt agaggatcga gatcgatctc gatcccgcga aattaatacg actcactata
7321 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
7381 gagatatacc atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac
7441 cgtagcgcag gccgctccga aagataacac ctggtacact ggtgctaaac tgggctggtc
7501 ccagtaccat gacactggtt tcatcaacaa caatggcccg acccatgaaa accaactggg
7561 cgctggtgct tttggtggtt accaggttaa cccgtatgtt ggctttgaaa tgggttacga
7621 ctggttaggt cgtatgccgt acaaaggcag cgttgaaaac ggtgcataca aagctcaggg
7681 cgttcaactg accgctaaac tgggttaccc aatcactgac gacctggaca tctacactcg
7741 tctgggtggc atggtatggc gtgcagacac taaatccaac gtttatggta aaaaccacga
7801 caccggcgtt tctccggt
//
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