| 质粒名称: | pC-HYG-YR |
|---|---|
| 出品公司: | addgene |
| 目录编号: | 105788 |
| 存储实验室: | Andrzej Dziembowski |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA5/FRT/TO |
| 载体出品公司: | Invitrogen |
| 空载体大小: | 4038 bp |
| 完整质粒大小: | 5589 bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | Hygromycin |
| 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 |
|
| 标签/融合蛋白: | TEV-ProteinA (C terminal on backbone) |
|
文献引用方法 |
|
| 材料和方法部分: |
pKK-TEV-ProteinA was a gift from Andrzej Dziembowski (Addgene plasmid # 105788 ; http://n2t.net/addgene:105788 ; RRID:Addgene_105788)
|
| 参考文献部分: | Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system. Szczesny RJ, Kowalska K, Klosowska-Kosicka K, Chlebowski A, Owczarek EP, Warkocki Z, Kulinski TM, Adamska D, Affek K, Jedroszkowiak A, Kotrys AV, Tomecki R, Krawczyk PS, Borowski LS, Dziembowski A. PLoS One. 2018 Mar 28;13(3):e0194887. doi: 10.1371/journal.pone.0194887. eCollection 2018. 10.1371/journal.pone.0194887 PubMed 29590189 |
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 pKK-TEV-ProteinA 5589 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expression of your protein of interest in fusion with two IgG
binding domains of Staphylococcus aureus protein A at the C-terminus
(cleavable by TEV) (PMID: 3507693, 10504710). Protein purification..
ACCESSION .
VERSION .
KEYWORDS pKK-TEV-ProteinA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5589)
AUTHORS Szczesny RJ, Kowalska K, Klosowska-Kosicka K, Chlebowski A, Owczarek
EP, Warkocki Z, Kulinski TM, Adamska D, Affek K, Jedroszkowiak A,
Kotrys AV, Tomecki R, Krawczyk PS, Borowski LS, Dziembowski A
TITLE Versatile approach for functional analysis of human proteins and
efficient stable cell line generation using FLP-mediated
recombination system.
JOURNAL PLoS One. 2018 Mar 28;13(3):e0194887. doi:
10.1371/journal.pone.0194887. eCollection 2018.
PUBMED 29590189
REFERENCE 2 (bases 1 to 5589)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5589
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(39..58)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 230..609
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 610..813
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 764..784
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
protein_bind 815..833
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 836..854
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 864..888
/label=LNCX
/note="Human CMV promoter, forward primer"
CDS 997..1017
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQG"
CDS 1058..1078
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQG"
CDS 1118..1291
/codon_start=1
/product="IgG-binding unit of Staphylococcus aureus protein
A"
/label=ProtA
/translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
AEAKKLNDAQAPK"
CDS 1295..1465
/codon_start=1
/product="IgG-binding unit of Staphylococcus aureus protein
A"
/label=ProtA
/translation="DNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLA
EAKKLNGAQAPK"
primer_bind complement(1536..1553)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 1542..1766
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(1899..1918)
/label=F1ori-R
/note="F1 origin, reverse primer"
protein_bind 2050..2097
/label=FRT
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
CDS 2105..3127
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
"
polyA_signal 3257..3378
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3294..3313)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 3348..3367
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(3427..3443)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3427..3443)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(3440..3462)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 3451..3467
/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)."
promoter complement(3475..3505)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3520..3541
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(3658..3675)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(3829..4417)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(3909..3928)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4588..5448)
/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 5211..5230
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(5449..5553)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
61 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
121 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
181 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
241 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
301 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
361 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
421 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
481 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
541 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
601 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
661 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
721 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
781 cgtgtacggt gggaggtcta tataagcaga gctctcccta tcagtgatag agatctccct
841 atcagtgata gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc ctggagacgc
901 catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct ccggactctg
961 gcgtttaaac ttaagcttgg taccgagctc ggatccgaaa acctgtactt ccaaggaacc
1021 ggtggtcacc tgtacaatcg atcctgcagg agctagcgag aatttgtatt ttcagggtga
1081 tatcaaaacc gcggctcttg cgcaacacga tgaagccgtg gacaacaaat tcaacaaaga
1141 acaacaaaac gcgttctatg agatcttaca tttacctaac ttaaacgaag aacaacgaaa
1201 cgccttcatc caaagtttaa aagatgaccc aagccaaagc gctaaccttt tagcagaagc
1261 taaaaagcta aatgatgctc aggcgccgaa agtagacaac aaattcaaca aagaacaaca
1321 aaacgcgttc tatgagatct tacatttacc taacttaaac gaagaacaac gaaacgcctt
1381 catccaaagt ttaaaagatg acccaagcca aagcgctaac cttttagcag aagctaaaaa
1441 gctaaatggt gctcaggcgc cgaaagtaga cgcgaattcc gcggggaagt caacctgact
1501 cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
1561 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
1621 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
1681 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
1741 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
1801 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
1861 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
1921 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
1981 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
2041 acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt
2101 ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
2161 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
2221 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
2281 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
2341 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
2401 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga
2461 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
2521 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
2581 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
2641 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
2701 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
2761 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
2821 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
2881 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
2941 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
3001 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
3061 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
3121 ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
3181 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
3241 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
3301 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
3361 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
3421 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
3481 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
3541 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
3601 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
3661 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
3721 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
3781 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
3841 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
3901 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
3961 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
4021 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
4081 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
4141 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
4201 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
4261 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
4321 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
4381 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
4441 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
4501 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
4561 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
4621 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
4681 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
4741 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
4801 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
4861 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
4921 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
4981 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
5041 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
5101 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
5161 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
5221 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
5281 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
5341 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
5401 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
5461 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
5521 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
5581 cgtcgacgg
//
