Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012344 | pCEP4-M2L | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pCEP4-M2L
- Antibiotic Resistance:
- Ampicillin
- Length:
- 12852 bp
- Type:
- Episomal/EBNA
- Replication origin:
- ori
- Selection Marker:
- Hygromycin
- Copy Number:
- Low Copy
- Promoter:
- CMV
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- IRES-F
- 3' Primer:
- EBV_rev
pCEP4-M2L 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.
pCEP4-M2L vector Sequence
LOCUS Exported 12852 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION used in the derivation of human iPS cells using non-integrating
episomal vectors; expresses cMyc and Lin28.
ACCESSION .
VERSION .
KEYWORDS pCEP4-M2L
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12852)
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 12852)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Science.
2009 Mar 26."
FEATURES Location/Qualifiers
source 1..12852
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 4..383
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 384..587
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 538..558
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 584..608
/label=LNCX
/note="Human CMV promoter, forward primer"
CDS 683..2002
/codon_start=1
/product="human c-Myc proto-oncogene"
/label=c-Myc
/translation="MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAP
SEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTE
LLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPN
PARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDS
LLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGS
PSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNR
KCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKA
TAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA"
misc_feature 2069..2642
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
misc_feature 2092..2644
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
primer_bind complement(2236..2253)
/label=IRES reverse
/note="IRES internal ribosome entry site, reverse primer.
Also called pCDH-rev"
primer_bind 2463..2482
/label=IRES-F
/note="IRES internal ribosome entry site, forward primer"
polyA_signal 3338..3472
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3362..3381)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 3416..3435
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(3615..3637)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
rep_origin 4195..5984
/label=oriP
/note="Epstein-Barr virus oriP replication origin (Yates et
al., 2000)"
CDS complement(6286..8211)
/codon_start=1
/product="Epstein-Barr nuclear antigen 1, also known as
EBNA-1"
/label=EBNA1
/note="crucial for latent viral infection, and for episomal
amplification of vectors containing the oriP origin (Okita
et al., 2013)"
/translation="MSDEGPGTGPGNGLGEKGDTSGPEGSGGSGPQRRGGDNHGRGRGR
GRGRGGGRPGAPGGSGSGPRHRDGVRRPQKRPSCIGCKGTHGGTGAGAGAGGAGAGGAG
AGGGAGAGGGAGGAGGAGGAGAGGGAGAGGGAGGAGGAGAGGGAGAGGGAGGAGAGGGA
GGAGGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAGGGAGAGGAGGAGGAGAGGAGAGG
GAGGAGGAGAGGAGAGGAGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAGGAGAG
GGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAGAGGAGAGGGGRGRGGSGGRGRG
GSGGRGRGGSGGRRGRGRERARGGSRERARGRGRGRGEKRPRSPSSQSSSSGSPPRRPP
PGRRPFFHPVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGPRGQGDGGR
RKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVFVYGGSKTSL
YNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLK
DAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDGDEGGDGDE
GEEGQE"
primer_bind complement(8646..8664)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 8732..8836
/gene="bla"
/label=AmpR promoter
CDS 8837..9697
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(9055..9074)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 9868..10456
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 10357..10376
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
promoter 10887..11032
/label=HSV TK promoter
/note="herpes simplex virus thymidine kinase promoter"
CDS 11075..12112
/codon_start=1
/product="hygromycin B phosphotransferase"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
MGEAN"
primer_bind complement(12110..12129)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 12154..12202
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"