| 质粒名称: | pSBtet-Hyg |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 60508 |
| 存储实验室: | Eric Kowarz |
|
载体骨架基本信息 |
|
| 载体名称: | pUC19 |
| 空载体大小: | 2021 bp |
| 完整质粒大小: | 7664 bp |
| 载体类型: | 哺乳细胞表达 ; Transposon |
| 筛选标记: | Hygromycin |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | Top10 |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 克隆方法: | 限制性内切酶 |
| 5’测序引物 | CCTGGAGCCAATTCCAACTCT |
| 3’测序引物 | CACTGCATTCTTGTTGTGGTT |
|
文献引用方法 |
|
| 材料和方法部分: |
pSBtet-Hyg was a gift from Eric Kowarz (Addgene plasmid # 60508 ; http://n2t.net/addgene:60508 ; RRID:Addgene_60508)
|
| 参考文献部分: | Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines. Kowarz E, Loescher D, Marschalek R. Biotechnol J. 2015 Feb 4. doi: 10.1002/biot.201400821. 10.1002/biot.201400821 PubMed 25650551 |
买家导航
pSBtet-Hyg载体质粒基本信息
pSBtet-Hyg质粒图谱载体图谱和pSBtet-Hyg载体序列质粒序列多克隆位点信息
pSBtet-Hyg质粒载体简介
pSBtet-Hyg质粒序列载体序列
LOCUS pSBtet-Hyg 7664 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION SB-transposon with inducible SfiI cloning site for GOI (contains
firefly luciferase) and constitutive expression of rtTA and
hygromycin resistance gene.
ACCESSION .
VERSION .
KEYWORDS pSBtet-Hyg
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7664)
AUTHORS Kowarz E, Loescher D, Marschalek R
TITLE Optimized Sleeping Beauty transposons rapidly generate stable
transgenic cell lines.
JOURNAL Biotechnol J. 2015 Feb 4. doi: 10.1002/biot.201400821.
PUBMED 25650551
REFERENCE 2 (bases 1 to 7664)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7664
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 295..607
/label=tight TRE promoter
/note="Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter"
protein_bind 303..321
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 339..357
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 374..392
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 410..428
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 446..464
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 481..499
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 517..535
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 580..604
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 680..689
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 686..2338
/codon_start=1
/product="firefly luciferase"
/label=luciferase
/note="synthetic luc2 version of the luciferase gene"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
polyA_signal 2374..2495
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2385..2404)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 2439..2458
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
CDS 3218..3961
/codon_start=1
/product="improved tetracycline-controlled transactivator"
/label=rtTA-Advanced
/note="In the Tet-On(R) system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription only in
the presence of tetracycline or doxycycline."
/translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
CDS 3968..4024
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 4025..5050
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
primer_bind complement(5105..5122)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 5111..5335
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(5661..5678)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5832..6420)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5912..5931)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6591..7451)
/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 7214..7233
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7452..7556)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 ggatccctat acagttgaag tcggaagttt acatacactt aagttggagt cattaaaact
61 cgtttttcaa ctactccaca aatttcttgt taacaaacaa tagttttggc aagtcagtta
121 ggacatctac tttgtgcatg acacaagtca tttttccaac aattgtttac agacagatta
181 tttcacttat aattcactgt atcacaattc cagtgggtca gaagtttaca tacactaagt
241 tcgactcctc tgcagaatgc ggcgatgttt cggtaagggg tccgctatct agacgagttt
301 actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg
361 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt
421 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta
481 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta
541 tgtcgattag gcgtgtacgg tgggcgccta taaaagcaga gctcgtttag tgaaccgtca
601 gatcgcctgg agccaattcc aactcttttg ccttatacct actttccgta ccacttccta
661 ccctcgaaag gcctctgagg ccaccatgga agatgccaaa aacattaaga agggcccagc
721 gccattctac ccactcgaag acgggaccgc cggcgagcag ctgcacaaag ccatgaagcg
781 ctacgccctg gtgcccggca ccatcgcctt taccgacgca catatcgagg tggacattac
841 ctacgccgag tacttcgaga tgagcgttcg gctggcagaa gctatgaagc gctatgggct
901 gaatacaaac catcggatcg tggtgtgcag cgagaatagc ttgcagttct tcatgcccgt
961 gttgggtgcc ctgttcatcg gtgtggctgt ggccccagct aacgacatct acaacgagcg
1021 cgagctgctg aacagcatgg gcatcagcca gcccaccgtc gtattcgtga gcaagaaagg
1081 gctgcaaaag atcctcaacg tgcaaaagaa gctaccgatc atacaaaaga tcatcatcat
1141 ggatagcaag accgactacc agggcttcca aagcatgtac accttcgtga cttcccattt
1201 gccacccggc ttcaacgagt acgacttcgt gcccgagagc ttcgaccggg acaaaaccat
1261 cgccctgatc atgaacagta gtggcagtac cggattgccc aagggcgtag ccctaccgca
1321 ccgcaccgct tgtgtccgat tcagtcatgc ccgcgacccc atcttcggca accagatcat
1381 ccccgacacc gctatcctca gcgtggtgcc atttcaccac ggcttcggca tgttcaccac
1441 gctgggctac ttgatctgcg gctttcgggt cgtgctcatg taccgcttcg aggaggagct
1501 attcttgcgc agcttgcaag actataagat tcaatctgcc ctgctggtgc ccacactatt
1561 tagcttcttc gctaagagca ctctcatcga caagtacgac ctaagcaact tgcacgagat
1621 cgccagcggc ggggcgccgc tcagcaagga ggtaggtgag gccgtggcca aacgcttcca
1681 cctaccaggc atccgccagg gctacggcct gacagaaaca accagcgcca ttctgatcac
1741 ccccgaaggg gacgacaagc ctggcgcagt aggcaaggtg gtgcccttct tcgaggctaa
1801 ggtggtggac ttggacaccg gtaagacact gggtgtgaac cagcgcggcg agctgtgcgt
1861 ccgtggcccc atgatcatga gcggctacgt taacaacccc gaggctacaa acgctctcat
1921 cgacaaggac ggctggctgc acagcggcga catcgcctac tgggacgagg acgagcactt
1981 cttcatcgtg gaccggctga agagcctgat caaatacaag ggctaccagg tagccccagc
2041 cgaactggag agcatcctgc tgcaacaccc caacatcttc gacgccgggg tcgccggcct
2101 gcccgacgac gatgccggcg agctgcccgc cgcagtcgtc gtgctggaac acggtaaaac
2161 catgaccgag aaggagatcg tggactatgt ggccagccag gttacaaccg ccaagaagct
2221 gcgcggtggt gttgtgttcg tggacgaggt gcctaaagga ctgaccggca agttggacgc
2281 ccgcaagatc cgcgagattc tcattaaggc caagaagggc ggcaagatcg ccgtgtaagg
2341 cctgtcaggc caagcttcca tcgatagaca tgataagata cattgatgag tttggacaaa
2401 ccacaacaag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
2461 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
2521 tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat
2581 gtggtaccta ggcgagaccc tgtctcacaa aataaagtaa gcccggactg agtgcggaaa
2641 ggcgggcctg gcgggtctgg tctccccatg cgggccacca gaggccctgc agccttcagt
2701 cgcttgaagg ggtaatggcg cttccactca caaacatggc ggacagagcg tgtgaacgag
2761 atgaacagcc cctcaaaaat atggccgccg aggctggacg gccgtgcccc agcagcaccg
2821 cctccgcgcc ccacgtgatc tctcgccggg cacagcgctg accgcggagg tccaaccgga
2881 agaatgtccg gattggacat tcggaagagg gcccgccttc cctggggaat ctctgcgcac
2941 gcgcagaacg cttcgaccaa tgaaaacaca ggaagccgtc cgcgcaaccg cgttgcgtca
3001 cttctgccgc ccctgtttca aggtatatag ccgtagacgg aacttcgcct ttctctcggc
3061 cttagcgcca tttttttggg tgagtgtttt ttggttcctg cgttgggatt ccgtgtacaa
3121 tccatagaca tctgacctcg gcacttagca tcatcacagc aaactaactg tagcctttct
3181 ctctttccct gtagaaacct ctgcacctga ggccaccatg tctagactgg acaagagcaa
3241 agtcataaac ggcgctctgg aattactcaa tggagtcggt atcgaaggcc tgacgacaag
3301 gaaactcgct caaaagctgg gagttgagca gcctaccctg tactggcacg tgaagaacaa
3361 gcgggccctg ctcgatgccc tgccaatcga gatgctggac aggcatcata cccacttctg
3421 ccccctggaa ggcgagtcat ggcaagactt tctgcggaac aacgccaagt cattccgctg
3481 tgctctcctc tcacatcgcg acggggctaa agtgcatctc ggcacccgcc caacagagaa
3541 acagtacgaa accctggaaa atcagctcgc gttcctgtgt cagcaaggct tctccctgga
3601 gaacgcactg tacgctctgt ccgccgtggg ccactttaca ctgggctgcg tattggagga
3661 acaggagcat caagtagcaa aagaggaaag agagacacct accaccgatt ctatgccccc
3721 acttctgaga caagcaattg agctgttcga ccggcaggga gccgaacctg ccttcctttt
3781 cggcctggaa ctaatcatat gtggcctgga gaaacagcta aagtgcgaaa gcggcgggcc
3841 ggccgacgcc cttgacgatt ttgacttaga catgctccca gccgatgccc ttgacgactt
3901 tgaccttgat atgctgcctg ctgacgctct tgacgatttt gaccttgaca tgctccccgg
3961 gtccggcgct actaacttca gcctgctgaa gcaggctggt gacgtcgagg agaatcctgg
4021 tcccatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
4081 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
4141 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
4201 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
4261 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
4321 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
4381 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
4441 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt
4501 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
4561 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
4621 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
4681 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
4741 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
4801 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
4861 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
4921 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
4981 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
5041 aaaggaatag ttcgaaggcc tgtcgtgaag cttggggatc aattctctag agctcgctga
5101 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
5161 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
5221 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
5281 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
5341 gaggcggaaa gaaccagctg ggcctactag ctactcggga ccccttaccg aaacatcgcc
5401 gcattctgca gaggagtcga gtgtatgtaa acttctgacc cactgggaat gtgatgaaag
5461 aaataaaagc tgaaatgaat cattctctct actattattc tgatatttca cattcttaaa
5521 ataaagtggt gatcctaact gacctaagac agggaatttt tactaggatt aaatgtcagg
5581 aattgtgaaa aagtgagttt aaatgtattt ggctaaggtg tatgtaaact tccgacttca
5641 actgtatagg gatccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
5701 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
5761 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
5821 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
5881 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
5941 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
6001 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
6061 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
6121 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
6181 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
6241 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
6301 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
6361 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
6421 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
6481 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
6541 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
6601 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
6661 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
6721 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
6781 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
6841 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
6901 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
6961 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
7021 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
7081 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
7141 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
7201 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
7261 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
7321 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
7381 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
7441 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
7501 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
7561 tttccccgaa aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa
7621 ataccgcatc aggaaattgt aagcgttaat attttgttaa aatt
//
