| 质粒名称: | piggyBac CMV-GFP-FRT |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 92078 |
| 存储实验室: | Jae Yong Han |
|
载体骨架基本信息 |
|
| 载体名称: | pCyL50 |
| 载体出品公司: | Sanger institute |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 3050 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达；piggyBac |
| 筛选标记: | 新霉素（G418） |
| 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: | CMV-eGFP and TK-Neo between FRT3 and FRT |
| 物种来源: | -- |
| 插入大小: | 4000 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | CMV |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | HindIII（销毁后未知） |
| 3' 克隆位点: | NotI（销毁后未知） |
| 5' 测序引物 | T3, M13R |
| 3' 测序引物 | T7, M13F(-20) |
|
文献引用方法 |
|
| 材料和方法部分: | piggyBac CMV-GFP-FRT was a gift from Jae Yong Han (Addgene plasmid # 92078 ; http://n2t.net/addgene:92078 ; RRID:Addgene_92078) |
| 参考文献部分: | Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange. Lee HJ, Lee HC, Kim YM, Hwang YS, Park YH, Park TS, Han JY. FASEB J. 2016 Feb;30(2):555-63. doi: 10.1096/fj.15-274712. Epub 2015 Oct 6. 10.1096/fj.15-274712 PubMed 26443821 |
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 piggyBac CMV-GFP-FRT 7077 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION production of transgenic animal and recombination using flipase.
ACCESSION .
VERSION .
KEYWORDS piggyBac CMV-GFP-FRT
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7077)
AUTHORS Lee HJ, Lee HC, Kim YM, Hwang YS, Park YH, Park TS, Han JY
TITLE Site-specific recombination in the chicken genome using Flipase
recombinase-mediated cassette exchange.
JOURNAL FASEB J. 2016 Feb;30(2):555-63. doi: 10.1096/fj.15-274712. Epub 2015
Oct 6.
PUBMED 26443821
REFERENCE 2 (bases 1 to 7077)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7077
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 381..584
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 535..555
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
promoter 629..647
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 686..695
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 692..1411
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(737..758)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(998..1017)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 1345..1366
/label=EGFP-C
/note="EGFP, forward primer"
promoter complement(1439..1457)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(1440..1457)
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind complement(1477..1494)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 1483..1707
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
protein_bind 1717..1764
/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)."
repeat_region 2114..2176
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
promoter complement(2243..2261)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2268..2285)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(2268..2284)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2277..2299)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 2426..2881
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2513..2532)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2723..2744
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 2907..3011
/gene="bla"
/label=AmpR promoter
CDS 3012..3872
/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 complement(3230..3249)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 4043..4631
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4532..4551
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4785..4802
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 4919..4940
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4955..4985
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4993..5009
/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)."
primer_bind 4998..5020
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 5017..5033
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 5017..5033
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
promoter 5054..5072
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
repeat_region 5221..5255
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
protein_bind 5598..5631
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS 5916..6719
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(5979..5998)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 6589..6608
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 6726..6774
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
protein_bind 6785..6818
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind complement(6887..6906)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
661 ggtaccgagc tcggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac
721 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
781 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
841 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
901 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
961 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
1021 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
1081 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
1141 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
1201 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
1261 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
1321 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
1381 cactctcggc atggacgagc tgtacaagta aagcggccgc gactctagag ggccctattc
1441 tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc
1501 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
1561 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
1621 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
1681 atgctgggga tgcggtgggc tctatggctt gtcgacgaag ttcctattcc gaagttccta
1741 ttctctagaa agtataggaa cttcctcgag ttaattaacg agagcataat attgatatgt
1801 gccaaagttg tttctgactg actaataagt ataatttgtt tctattatgt ataggttaag
1861 ctaattactt attttataat acaacatgac tgtttttaaa gtacaaaata agtttatttt
1921 tgtaaaagag agaatgttta aaagttttgt tactttatag aagaaatttt gagtttttgt
1981 ttttttttaa taaataaata aacataaata aattgtttgt tgaatttatt attagtatgt
2041 aagtgtaaat ataataaaac ttaatatcta ttcaaattaa taaataaacc tcgatataca
2101 gaccgataaa acacatgcgt caattttacg catgattatc tttaacgtac gtcacaatat
2161 gattatcttt ctagggttaa ataatagttt ctaatttttt tattattcag cctgctgtcg
2221 tgaataccga gctccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
2281 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
2341 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
2401 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
2461 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
2521 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
2581 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
2641 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
2701 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
2761 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
2821 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
2881 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
2941 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
3001 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
3061 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
3121 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
3181 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
3241 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
3301 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
3361 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
3421 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
3481 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
3541 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
3601 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
3661 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
3721 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
3781 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
3841 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
3901 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
3961 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
4021 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
4081 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
4141 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
4201 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
4261 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
4321 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
4381 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
4441 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
4501 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
4561 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
4621 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
4681 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
4741 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
4801 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
4861 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
4921 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
4981 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
5041 acgccaagct cgaaattaac cctcactaaa gggaacaaaa gctggtacct cgcgcgactt
5101 ggtttgccat tctttagcgc gcgtcgcgtc acacagcttg gccacaatgt ggtttttgtc
5161 aaacgaagat tctatgacgt gtttaaagtt taggtcgagt aaagcgcaaa tcttttttaa
5221 ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt gctctctctt
5281 tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg cttggagctc
5341 ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata acgaccgcgt
5401 gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact catacgataa
5461 ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata ttttcttgtt
5521 atagatatcg aagttcctat tccgaagttc ctattcttca aatagtatag gaacttcgtc
5581 gagatcgatg gcgcgccata acttcgtata gcatacatta tacgaagtta tgaatttcga
5641 gcagtgtggt tttcaagagg aagcaaaaag cctctccacc caggcctgga atgtttccac
5701 ccaatgtcga gcagtgtggt tttgcaagag gaagcaaaaa gcctctccac ccaggcctgg
5761 aatgtttcca cccaatgtcg agcaaacccc gcccagcgtc ttgtcattgg cgaattcgaa
5821 cacgcagatg cagtcggggc ggcgcggtcc caggtccact tcgcatatta aggtgacgcg
5881 tgtggcctcg aacaccgagc gaccctgcag ccaatatggg atcggccatt gaacaagatg
5941 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
6001 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
6061 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
6121 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
6181 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
6241 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
6301 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
6361 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
6421 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg
6481 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
6541 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
6601 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
6661 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
6721 gggatcggca ataaaaagac agaataaaac gcacgggtgt tgggtcgttt gttcggatca
6781 attcataact tcgtatagca tacattatac gaagttatgg cgcgccttaa ttaaggatcc
6841 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
6901 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
6961 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
7021 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
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