Cart 2

pEP4 E02S ET2K vector (V000770) Gene synthesis in pEP4 E02S ET2K backbone

Price Information

Cat No. Plasmid Name Availability Buy one, get one free! (?)
V000770 pEP4 E02S ET2K In stock, instant shipping

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

Copy Sequence View Map
pEP4 E02S ET2K17522 bp80016002400320040004800560064007200800088009600104001120012000128001360014400152001600016800EF-1-alpha core promoterEF-1-alpha intron AhOct4IREShSOX2SV40 poly(A) signalEF-1-alpha promoterlarge T antigenIRES2hKLF4oriPEBNA1pBRforEcoAmpR promoterAmpRori

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"