| 质粒名称: | pCD5-H3TX12-CO-GCN4-TEV-sfGFP-TS |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 158739 |
| 存储实验室: | Robert de Vries |
|
载体骨架基本信息 |
|
| 载体名称: | pCD5 |
| 空载体大小: | 5400 bp |
| 完整质粒大小: | 6935 bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | Ampicillin and Tetracycline |
| 生长温度: | 37 ℃ |
| 宿主菌: | MC1061 |
| 拷贝数: | 低拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | Hemagglutinin A/TX/50/12 H3 |
| 插入大小: | 1525 bp |
| 基因突变: | G186V S219F |
| GenBank编号 | AGL06219.1 |
| 启动子: | CMV |
| 标签/融合蛋白: | GCN4-TEV-sfGFP-TwinStrep (C terminal on insert) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | NheI(未破坏) |
| 3’克隆位点: | PacI(未破坏) |
| 5’测序引物 | GGC TAA CTA GAG AAC CCA CTG C |
| 3’测序引物 | TTC AGG GGG AGG TGT GGG AG |
|
文献引用方法 |
|
| 材料和方法部分: |
pCD5-H3TX12-CO-GCN4-TEV-sfGFP-TS was a gift from Robert de Vries (Addgene plasmid # 158739 ; http://n2t.net/addgene:158739 ; RRID:Addgene_158739)
|
| 参考文献部分: | Drivers of recombinant soluble influenza A virus hemagglutinin and neuraminidase expression in mammalian cells. van der Woude R, Turner HL, Tomris I, Bouwman KM, Ward AB, de Vries RP. Protein Sci. 2020 Jul 25. doi: 10.1002/pro.3918. 10.1002/pro.3918 PubMed 32710576 |
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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 pCD5-H3TX12-CO-GCN4-TEV-sfGFP-TS 5501 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses influenza A virus trimeric hemagglutinin A/H3/TX/12 fused
to a sfGFP.
ACCESSION .
VERSION .
KEYWORDS pCD5-H3TX12-CO-GCN4-TEV-sfGFP-TS
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5501)
AUTHORS van der Woude R, Turner HL, Tomris I, Bouwman KM, Ward AB, de Vries
RP
TITLE Drivers of recombinant soluble influenza A virus hemagglutinin and
neuraminidase expression in mammalian cells.
JOURNAL Protein Sci. 2020 Jul 25. doi: 10.1002/pro.3918.
PUBMED 32710576
REFERENCE 2 (bases 1 to 5501)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5501
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(749..769)
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQA"
CDS complement(776..793)
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS complement(2099..2125)
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
enhancer 2923..3302
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
tRNA 3475..3654
/product="E. coli amber-suppressor tyrosine tRNA"
/label=supF
rep_origin 3777..4208
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3864..3883)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4077..4098
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4320..4339)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
promoter complement(4820..5149)
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 4834..4969
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind complement(4888..4907)
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind 5134..5154
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
polyA_signal 5189..5310
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5200..5219)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 5254..5273
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
CDS complement(5458..46)
/codon_start=1
/product="two Strep-Tag(R)II moieties connected by a linker
for enhanced binding to Strep-Tactin(R), an engineered form
of streptavidin (Schmidt et al., 2013)"
/label=Twin-Strep-tag
/translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
ORIGIN
1 ccccagaccc cccccccttc tcaaactgtg ggtggctcca ggcgctgctc attccgtgtg
61 tgatgcctgc tgcggtcacg aactccagca gcaccatgtg atccctcttc tcgtttgggt
121 ccttggacag cacgctctgt gtagacagat agtgattatc aggcagcagc acgggtccgt
181 cgccgatagg ggtgttctgc tggtagtgat cggccagctg cacagagccg tcctccacat
241 tgtgtctgat cttgaagttg gccttgatgc cgttcttctg cttatcggcg gtgatataca
301 cgttgtggga attaaagttg tactccagct tgtggcccag gatattgcca tcctccttga
361 agtcgatgcc cttcagctcg atccggttca ccagtgtgtc gccctcaaac ttcacctcgg
421 cgcgtgtctt ataggtgcca tcgtccttga agctgatggt cctctcctgc acgtagccct
481 cgggcatggc agacttaaag aaatcgtgtc tcttcatgtg gtctggatac cgggagaaac
541 actgcacgcc gtatgtcagt gtggtcacca gggttggcca aggcacgggc agcttgcctg
601 tggtgcagat aaacttcagt gtcagcttgc cgttggttgc gtcgccctct ccctctcctc
661 tcacgctgaa cttgtggcca ttgacatcgc catccagttc caccaggata gggaccacgc
721 cagtaaacag ttcttcgcct ttgctcatag cctgaaaata cagattctcg tcgacgctgc
781 cgcgcggcac cagcttaatc ttcttgatgc gggcgatctc gttctcgatc ttcttctgct
841 tggactcgat ctcttcgatc ttgtcctcga tctgcttcat gcgcttaatt aacttcagct
901 ccacgccctt gatctgaaac cgattgttca gggcctcgtc gcggtacacg tcgtggtcgt
961 atgtgccgtt cctgatgctg ccgatacagg cattatcgca cttgtggtag atcttaaagc
1021 agccattgcc catgtcctcg gcgttctctc tcagctgctt cttggtcttc tcgaacagct
1081 tgttcatctc ggaatctgtc aggtcgatgg tgtgctggtt ctccagggcc accagcagct
1141 cggcattata gctccacaga tcgatcttgg tgtcctccac gtacttctcc agatcctgga
1201 tccggccctc cacctcggaa aactccttct cgatctggtg gaacttctca tttgtcttgc
1261 cgatcaggcg attcagcttg ccgttgatct gatcgatggc ggcctgggtg ctcttcaggt
1321 ctgctgcctg tccccttccc tcagagttct ggtgtctgaa gccataccag ccgtccacca
1381 tgccctccca gccattctcg ataaagcctg cgattgctcc gaagattccc ctggtctgct
1441 tctctggcac gttcctcatg cctgtggcca gcttcagggt agactgcttc acatatcttg
1501 gacaggcgcc gtatgtgatt ctattcacgt tctgaaaggg cttatcattt gggatggagc
1561 cgttaggggt gatacactcg ctcttgcact tgccgatggg ggcgtcagac cgcatgatgg
1621 agctcttgcc ggacctgatc ttgaagtagc ctcttggggc gatcagattg cctgtggagt
1681 tgatcagcag gatatcgcca ggcttcacga tggtccaata gatagagatg cgggagggga
1741 tgttccggat gcgtggccta gagccgatat tagggatcac ggcctgctgg ctccgctttg
1801 tagacacggt gatccttccg gatggctgtg cgtacaggaa gatctgatcc ttgtctgtgc
1861 ctgggtggtg cacgccccag atatacagct tgtcaaactg ctcattgttt ggcattgtca
1921 cgttcagggc agggtacttg aaattcaggt gggtcagcca gttcagcctg ctaaagaaag
1981 aattgttgga tctccggatg caggcagagg atgtgccatt ctgggtcacg ccattccagt
2041 taaaggactc attgttgaac tccagggtgc cgctagaggc caccaggctc ctcagagagg
2101 cataatcagg cacgtcatag gggtaacagt tggagtaggc cttgcttctc tccacaaaca
2161 gatcccactt cttattctgg aagccatcgc actgtgggtc gcccagcagt gcgtcgatca
2221 gtgtacagtt ctcgccatcc aggatctggt gagggctgtc gcagatctcg ccgatggagc
2281 tgttctgcac cagctctgtg gcattggtca cctcgattct atcgtttgtg atggtcttca
2341 cgatggtgcc attaggcact gcgtggtgtc ccaggcacag ggtggcggta gaattgtcgt
2401 tgccgggcag cttctgagct agcacggaag cgaccagcat ccccagcagg tacaaggtgg
2461 ccagcggttg cagagacccc atgggcatgg tttctggcct tctcttgcct cagcctgggc
2521 agctgggcac cgcaggtgag ggtgtctggc cgggtatctc ctttagagca caatggatct
2581 cgaggtcgag ggatctctag aagctttatt gaggcttaag cagtgggttc tctagttagc
2641 cagagagctc ccaggctcag atctggtcta accagagaga cccagtacag gcaaaaagca
2701 gctgcttata tgcaggatct gagggctcgc cactccccag tcccgcccag gaattccgcc
2761 catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg
2821 ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa
2881 ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac
2941 taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg
3001 ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac
3061 ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa
3121 agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc
3181 gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata
3241 tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg
3301 tcaacgcgta tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag
3361 attcttcatg caattgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca
3421 ctactcagcg acctccaaca cacaagcagg gagcagcttt ctcaacgtaa cactttacag
3481 cggcgcgtca tttgatatga tgcgccccgc ttcccgataa gggagcaggc cagtaaaagc
3541 attacctgtg gtggggttcc cgagcggcca aagggagcag actctaaatc tgccgtcatc
3601 gacttcgaag gttcgaatcc ttcccccacc accatcactt tcaaaagtcc gaaagaccgc
3661 ggtccagctg gcgtaccgtt cctgtctaaa atccctttaa tcggcctcct gtttagctcc
3721 cgctctgatt ctaacgagga aagcacgtta tacgtgctcg tcaaagcaac catagtacgc
3781 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
3841 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
3901 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
3961 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggcggcc
4021 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
4081 actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata
4141 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
4201 cgcgaatttt aacaaaatct agcttgcgtt gctggcgttt ttccataggc tccgcccccc
4261 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
4321 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
4381 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
4441 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
4501 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatctcttga gtccaacccg
4561 gtaagacacg acttatcgcc actggcagca gccactggta actggcttca gcagagcgag
4621 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
4681 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
4741 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
4801 gattacgccg aattagcttt ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact
4861 tctggaatag ctcagaggcc gaggcggcct cggcctctgc ataaataaaa aaaattagtc
4921 agccatgggg cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta
4981 ggggcgggac tatggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
5041 gggagcctgg ggactttcca cacctggttg ctgactaatt gagatgcatg ctttgcatac
5101 ttctgcctgc tggggagcct ggggactttc cacaccctaa ctgacacaca ttccacagga
5161 tcggaagatc gatcttcccc agacatgata agatacattg atgagtttgg acaaaccaca
5221 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
5281 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
5341 caggttcagg gggaggtgtg ggaggttttt taaatctctg taggtagttt gtccaattat
5401 gtcacaccac agaagtaagg ttccttcaca aagatcctct agagtcgcgg ccgctcactt
5461 ttcaaactga ggatgtgacc aagcagaccc cccactcccc c
//
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