Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000770 | pEP4 E02S ET2K | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pEP4-E02S-ET2K is an episomal vector used in the derivation of human iPS cells without integrating into the host genome. It expresses the transcription factors Oct4 and Sox2, as well as SV40 large T antigen (SV40LT) and Klf4, which are crucial for the reprogramming process.
- Vector Name:
- pEP4 E02S ET2K
- Antibiotic Resistance:
- Ampicillin
- Length:
- 17522 bp
- Type:
- Episomal/EBNA
- Replication origin:
- ori
- Copy Number:
- Low Copy
- Promoter:
- EF-1α
- Cloning Method:
- Restriction Enzyme
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
pEP4 E02S ET2K vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
References
- Yu, Junying et al. “Human induced pluripotent stem cells free of vector and transgene sequences.” Science (New York, N.Y.) vol. 324,5928 (2009): 797-801. doi:10.1126/science.1172482
pEP4 E02S ET2K vector Sequence
LOCUS 40924_17577 17522 bp DNA circular SYN 13-MAY-2021
DEFINITION used in the derivation of human iPS cells using non-integrating
episomal vectors; expresses Oct4 and Sox2; SV40LT and Klf4 .
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 17522)
AUTHORS Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA
TITLE Human Induced Pluripotent Stem Cells Free of Vector and Transgene
Sequences
JOURNAL Science. 2009 Mar 26.
PUBMED 19325077
REFERENCE 2 (bases 1 to 17522)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 17522)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Science.
2009 Mar 26."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..17522
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 32..243
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
intron 244..1166
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
CDS 1229..2308
/codon_start=1
/label=hOct4
/note="Homo sapiens Oct-4 gene. Encodes a transcription
factor containing a POU homeodomain that plays a key role
in embryonic development and stem cell pluripotency.
Aberrant expression in adult tissues is associated with
tumorigenesis."
/translation="MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGP
GIGPGVGPGSEVWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAXVGVE
SNSDGASPEPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYT
QADVGLTLGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICK
AETLVQARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNR
RQKGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFP
EGEAFPPVSVTTLGSPMHSN"
misc_feature 2385..2958
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 2991..3941
/codon_start=1
/label=hSOX2
/note="Homo sapiens transcription factor SOX-2 gene.
Belongs to the SRY-related HMG-box (SOX) family of
transcription factors, which is involved in the regulation
of embryonic development and in the determination of cell
fate"
/translation="MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPM
NAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKE
HPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHM
NGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPT
YSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGA
EVPEPAAPSRLHMSQHYQSGPVPGTAINGTLPLSHM"
polyA_signal complement(3977..4111)
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
promoter 4641..5803
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 5946..8069
/codon_start=1
/label=large T antigen
/note="large T antigen from SV40 (simian virus 40)"
/translation="MDKVLNREESLQLMDLLGLERSAWGNIPLMRKAYLKKCKEFHPDK
GGDEEKMKKMNTLYKKMEDGVKYAHQPDFGGFWDATEIPTYGTDEWEQWWNAFNEENLF
CSEEMPSSDDEATADSQHSTPPKKKRKVEDPKDFPSELLSFLSHAVFSNRTLACFAIYT
TKEKAALLYKKIMEKYSVTFISRHNSYNHNILFFLTPHRHRVSAINNYAQKLCTFSFLI
CKGVNKEYLMYSALTRDPFSVIEESLPGGLKEHDFNPEEAEETKQVSWKLVTEYAMETK
CDDVLLLLGMYLEFQYSFEMCLKCIKKEQPSHYKYHEKHYANAAIFADSKNQKTICQQA
VDTVLAKKRVDSLQLTREQMLTNRFNDLLDRMDIMFGSTGSADIEEWMAGVAWLHCLLP
KMDSVVYDFLKCMVYNIPKKRYWLFKGPIDSGKTTLAAALLELCGGKALNVNLPLDRLN
FELGVAIDQFLVVFEDVKGTGGESRDLPSGQGINNLDNLRDYLDGSVKVNLEKKHLNKR
TQIFPPGIVTMNEYSVPKTLQARFVKQIDFRPKDYLKHCLERSEFLLEKRIIQSGIALL
LMLIWYRPVAEFAQSIQSRIVEWKERLDKEFSLSVYQKMKFNVAMGIGVLDWLRNSDDD
DEDSQENADKNEDGGEKNMEDSGHETGIDSQSQGSFQAPQSSQSVHDHNQPYHICRGFT
CFKKPPTPPPEPET"
misc_feature 8163..8746
/label=IRES2
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 8772..10181
/codon_start=1
/label=hKLF4
/note="Homo sapiens Kruppel-like factor 4 (Klf4) gene.
Belongs to the relatively large family of SP1-like
transcription factors and is involved in the regulation of
proliferation, differentiation, apoptosis and somatic cell
reprogramming."
/translation="MAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRL
PPVLPGRPYDLAAATVATDLESGGAGAACGGSNLAPLPRRETEEFNDLLDLDFILSNSL
THPPESVAATVSSSASASSSSSPSSSGPASAPSTCSFTYPIRAGNDPGVAPGGTGGGLL
YGRESAPPPTAPFNLADINDVSPSGGFMAELLRPELDPVYIPPQQPQPPGGGLMGKFVL
KASLSAPGSEYGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGA
GPPLSNGHRPAAHDFPLGRQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYP
SFLPDQMQPQVPPLHYQELMPPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYT
KSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSR
SDHLALHMKRHF"
promoter 10013..11175
/label=EF1A
/note="Human eukaryotic translation elongation factor 1
alpha-1 promoter"
rep_origin complement(11088..12877)
/direction=LEFT
/label=oriP
/note="Epstein-Barr virus oriP replication origin (Yates et
al., 2000)"
CDS complement(13182..15104)
/codon_start=1
/label=EBNA1
/note="Epstein-Barr nuclear antigen 1, also known as
EBNA-1"
/translation="MSDEGPGTGPGNGLGEKGDTSGPEGSGGSGPQRRGGDNHGRGRGR
GRGRGGGRPGAPGGSGSGPRHRDGVRRPQKRPSCIGCKGTHGGTGAGAGAGGAGAGGAG
AGGGAGAGGGAGGAGGAGGAGAGGGAGAGGGAGGAGGAGAGGGAGAGGGAGGAGAGGGA
GGAGGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAGGGAGAGGAGGAGGAGAGGAGAGG
GAGGAGGAGAGGAGAGGAGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAGGAGAG
GGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAGAGGAGAGGGGRGRGGSGGRGRG
GSGGRGRGGSGGRRGRGRERARGGSRERARGRGRGRGEKRPRSPSSQSSSSGSPPRRPP
PGRRPFFHPVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGPRGQGDGGR
RKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVFVYGGSKTSL
YNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLK
DAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDGDEGGDGDE
GEEGQE"
primer_bind complement(15543..15561)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 15629..15733
/label=AmpR promoter
CDS 15734..16591
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 16765..17353
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"