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pNiFty3-I-SEAP vector (V014484)

Price Information

Cat No. Plasmid Name Availability Add to cart
V014484 pNiFty3-I-SEAP In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
pNiFty3-I-SEAP
Antibiotic Resistance:
Bleomycin
Length:
4571 bp
Type:
Protein expression
Replication origin:
ori
Host:
Mammalian cells
Selection Marker:
Zeo
Promoter:
EF-1α core
Growth Temperature:
30℃

pNiFty3-I-SEAP vector Map

Copy Sequence View Map
pNiFty3-I-SEAP4571 bp600120018002400300036004200poly(A) signalpause siteSEAPSV40 poly(A) signalbeta-globin poly(A)BleoREM7 promoter5' LTR (truncated)EF-1-alpha core promoterori

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.

pNiFty3-I-SEAP vector Sequence

LOCUS       62056_18030        4571 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4571)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4571
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1..49
                     /label=poly(A) signal
                     /note="synthetic polyadenylation signal"
     misc_feature    60..151
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from
                     the human alpha-2 globin gene"
     CDS             636..2153
                     /codon_start=1
                     /label=SEAP
                     /note="secreted alkaline phosphatase from human placenta"
                     /translation="VLGPCMLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAA
                     KKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYVALSKT
                     YNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGK
                     SVGVVTTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVIL
                     GGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLD
                     PSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHG
                     HHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGL
                     APGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGED
                     VAVFARGPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGTTD"
     polyA_signal    complement(2216..2337)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    complement(2437..2831)
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     CDS             complement(2848..3219)
                     /codon_start=1
                     /label=BleoR
                     /note="antibiotic-binding protein"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(3239..3286)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     LTR             complement(3316..3584)
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human
                     T-cell leukemia virus (HTLV) type 1"
     promoter        complement(3597..3808)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     rep_origin      complement(3876..4464)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"