Human P4HB ORF clone (NM_000918) (Cat. No.: V035890)

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Basic Information

Name:: pCMV-Neo-P4HB(human)-mScarlet-2I

Accession ID:: NM_000918.4

Antibiotic Resistance:: Kanamycin

Length:: 6213 bp

Type:: Protein expression

Replication origin:: ori

Host:: Mammalian cells

Selection Marker:: Neo/G418;mScarlet-2I

Promoter:: CMV

3' Primer:: Sv40-polyA-R

Fusion Tag:: mScarlet-2I

Growth Strain(s):: DH5a

Growth Temperature:: 37℃

Expression Method:: Transient

Gene Synonyms:: CLCRP1; DSI; ERBA2L; GIT; P4Hbeta; PDI; PDIA1; PHDB; PO4DB; PO4HB; PROHB

Transcript Definition:: Homo sapiens prolyl 4-hydroxylase subunit beta (P4HB), mRNA

Cat. No.: V035890 pCMV-Neo-P4HB(human)-mScarlet-2I

$ 298.2

In stock, 1-2 weeks for quality controls

Two tubes of transfection grade plasmid, each tube is about 100 µg (lyophilized).

Resources

Transcript ID

NM_000918.4

Fusion Tag

mScarlet-2I 3xFLAG Myc-6xHis

Selection Marker

Neo/G418;mScarlet-2I Puro Puro;CopGFP Neo/G418

Expression Method

Transient Constiutive, Stable / Transient

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.

Human P4HB ORF clone (NM_000918) (Cat. No.: V035890) Sequence

LOCUS       V035890                 6213 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V035890
VERSION     V035890
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     promoter        join(5956..6213,1..100)
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             135..929
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /note="confers resistance to neomycin, kanamycin, and G418
                     (Geneticin®)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /transl_table=1
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
                     /label="NeoR/KanR"
     polyA_signal    1161..1208
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      1537..2125
                     /direction=RIGHT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     enhancer        2300..2603
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        2604..2807
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     CDS             2852..3538
                     /codon_start=1
                     /product="bright cysteine-free monomeric red fluorescent
                     protein with fast and complete maturation (Gadella et al.,
                     2023)"
                     /transl_table=1
                     /translation="MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LRVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGA
                     VSVAQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKM
                     ALRLKDGGRYLADFKTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHS
                     TGGSGGS"
                     /label="mScarlet3"
     CDS             3584..5107
                     /label="P4HB(NM_000918)"
                     /note="P4HB(NM_000918)"
                     /gene="P4HB"
     polyA_signal    5240..5361
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      complement(5368..5823)
                     /direction=LEFT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        5850..5954
                     /gene="bla"
                     /label="AmpR promoter"