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pCE-hSK vector (V010610) Gene synthesis in pCE-hSK backbone

Price Information

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V010610 pCE-hSK In stock, instant shipping

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Vector Name:
pCE-hSK
Antibiotic Resistance:
Ampicillin
Length:
11876 bp
Type:
Mammalian Expression
Replication origin:
ori
Copy Number:
High Copy
Promoter:
CAG
Cloning Method:
Restriction Enzyme
5' Primer:
pCAG-F
3' Primer:
TTAGCCAGAAGTCAGATGCTC
Growth Strain(s):
DH10B
Growth Temperature:
37℃

pCE-hSK vector Map

Copy Sequence View Map
pCE-hSK11876 bp50010001500200025003000350040004500500055006000650070007500800085009000950010000105001100011500CMV enhancerchicken beta-actin promoterchimeric intronpCAG-FhSOX2F2AhKLF4Bglob-pA-Rbeta-globin poly(A) signalIn lacZ genelac promoterCAP binding siteEBNA1oriPL4440oriAmpRAmpR promoter

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

  • Okita K, Yamakawa T, Matsumura Y, Sato Y, Amano N, Watanabe A, Goshima N, Yamanaka S. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells. 2013 Mar;31(3):458-66. doi: 10.1002/stem.1293. PMID: 23193063.

pCE-hSK vector Sequence

LOCUS       pCE-hSK       11876 bp DNA     circular SYN 20-JUL-2025
DEFINITION  Non-integrating (episomal) expression of human SOX2 and KLF4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11876)
  AUTHORS   Okita K, Yamakawa T, Matsumura Y, Sato Y, Amano N, Watanabe A, 
            Goshima N, Yamanaka S
  TITLE     An Efficient Non-viral Method to Generate Integration-Free Human iPS
            Cells from Cord Blood and Peripheral Blood Cells.
  JOURNAL   Stem Cells. 2012 Nov 29. doi: 10.1002/stem.1293.
  PUBMED    23193063
REFERENCE   2  (bases 1 to 11876)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 11876)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 11876)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Stem Cells.
            2012 Nov 29. doi: 10.1002/stem.1293."
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..11876
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..658
                     /label=chicken beta-actin promoter
     intron          659..1675
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1683..1702
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids,
                     forward primer"
     CDS             1740..2690
                     /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"
     CDS             2715..2780
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             2805..4214
                     /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
                     YGRESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVL
                     KASLSAPGSEYGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGA
                     GPPLSNGHRPAAHDFPLGRQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYP
                     SFLPDQMQPQVPPLHYQELMPPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYT
                     KSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSR
                     SDHLALHMKRHF"
     primer_bind     complement(4320..4339)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4385..4440
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4439..4458)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also
                     called rb-glob-pA-term-R"
     primer_bind     complement(4801..4817)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4801..4817)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4814..4836)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4825..4841
                     /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)."
     promoter        complement(4849..4879)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4894..4915)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             5394..7316
                     /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"
     rep_origin      7621..9410
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
     primer_bind     complement(9951..9968)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10122..10710)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(10881..11741)
                     /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     11504..11523
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11742..11846)
                     /label=AmpR promoter