Puc57 vector

Manufactured by Sangon

Sourced in China

The PUC57 vector is a plasmid commonly used for cloning and expression in Escherichia coli. It contains the origin of replication (ori) and the ampicillin resistance gene (bla), allowing for selection and maintenance of the plasmid in bacterial cells.

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Lab products found in correlation

Human hek293t, by American Type Culture Collection (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Pcdna3.1 vector, by Sangon (1 mentions) Small interfering rnas sirna, by GenePharma (1 mentions) Minibest plasmid purification kit, by Takara Bio (1 mentions) Abi quantstudio 3, by Thermo Fisher Scientific (1 mentions) Sybr premix ex taqtm tli rnaseh plus, by Takara Bio (1 mentions) Taqman genotyping master mix, by Thermo Fisher Scientific (1 mentions) Ecori, by Takara Bio (1 mentions) Bamhi, by Takara Bio (1 mentions) Bamh 1, by Sangon (1 mentions) Ecori, by Sangon (1 mentions)

Spelling variants of this product

PUC57 (9 citations) PUC57 plasmid vector (3 citations)

14 protocols using puc57 vector

Lentiviral Overexpression of HRP3 in HepG2 Cells

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HepG2 (ATCC HB-8065) and HEK293T human embryonic kidney cells (ATCC #CRL-11268) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. Stable overexpression of wild-type or mutant HRP3 in HepG2 cells was performed using FUGW lentiviral constructs expressing wild-type or mutant HRP3. Lentiviral vector infections (47 (link)) and plasmid transfections (48 (link)) were performed as previously described. Overexpression efficiency was determined by western blot.
The lentiviral vector containing wild-type HRP3 was constructed by inserting the HRP3 AgeI/EcoRI fragment amplified from the Puc57 vector (Sangon Biotech) into the AgeI/EcoRI-digested FUGW vector (Addgene). HRP3 mutants were generated by two-step PCR mutagenesis. All constructs were verified by sequencing.

Tian W., Yan P., Xu N., Chakravorty A., Liefke R., Xi Q, & Wang Z. (2019). The HRP3 PWWP domain recognizes the minor groove of double-stranded DNA and recruits HRP3 to chromatin. Nucleic Acids Research, 47(10), 5436-5448.

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Molecular Cloning of lncNSPL, RIG-I, and TRIM25 Constructs

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Sequence of lncNSPL (Gene number: 105370355,accession: XR_007083671), RIG-I and TRIM25 genes were obtained from the NCBI database. The lncNSPL plasmid construction process is divided into two steps. In the first step, the full length of lncNSPL was synthesized and cloned into pUC57 vector (Sangon Biotech, Shanghai, China). And then the full-length lncNSPL was cloned into the pcDNA3.1 eukaryotic expression vector and the retroviral vector GV367 by molecular cloning techniques, respectively. In this retroviral vector construct, lncNSPL transcribed was from RNA Pol II promoter of retroviral vectors GV367. Coding sequences of RIG-I and TRIM25 were cloned into pCDNA3.1 vector containing HA and Flag tags, respectively (Sangon Biotech, Shanghai). HA-tagged truncated structures of RIG-I were generated by standard molecular biology methods. Myc-Ub-K63 structure was a gift from Dr. Q. Zhang (Zhejiang University, China). Full-length lncNSPL was cloned in retroviral vectors GV367 for stable expression in THP-1 cells. LV10 lentiviral vector expressing control shRNA against RFP or shRNA targeting lncNSPL were obtained from Gene Pharma (Shanghai, China).
Small interfering RNAs (siRNAs) were designed and synthesizeds by GenePharma (Shanghai, China). The transfection of plasmids or siRNAs were performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions.

Jiang J., Li Y., Sun Z., Gong L., Li X., Shi F., Yao J., Meng Y., Meng X., Zhang Q., Wang Y., Su X, & Diao H. (2022). LncNSPL facilitates influenza A viral immune escape by restricting TRIM25-mediated K63-linked RIG-I ubiquitination. iScience, 25(7), 104607.

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Comprehensive V. parahaemolyticus Serotype Profiling

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A total of 244 reference bacterial isolates were used for the initial development and evaluation of the MLMA assay. This comprised of reference isolates (n = 176) representing 11 clinically most common V. parahaemolyticus serotypes during a 10-year period (2007–2017) from the Shenzhen Center for Disease Control and Prevention isolates collection (Table 1); and reference isolates (n = 66) representing additional 38 serotypes for detecting cross reactions (Table S1). For completeness, two rare serogroups absent during the 10 year period (O7 and O12) were incorporated into the assay using Escherichia coli TOP10 strains (n = 2) containing serogroup-specific genes (O7-wvcN, O12-wvcP) cloned into a pUC57 vector (Sangon Biotech Co. Ltd., Shanghai, China).

Li M., Jiang Y., Shi X., Li Y., Jiang M., Lin Y., Qiu Y., Zuo L., Deng Y., Lin Z., Liao Y., Li Q, & Hu Q. (2019). Simultaneous Identification of Clinically Common Vibrio parahaemolyticus Serotypes Using Probe Melting Curve Analysis. Frontiers in Cellular and Infection Microbiology, 9, 385.

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SARS-CoV-2 N Gene Quantification Protocol

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Targeted N gene fragments of SARS-CoV-2 were amplified by the primers same as used in the real-time RT-RAA assay and then cloned into PUC57 vector (Sangon Biotech, China). The concentration of this plasmid was measured after extraction (MiniBEST Plasmid Purification Kit TaKaRa, China). The expected copy number of N gene was calculated according to a previous study (Min et al., 2006 (link)).

Wu T., Ge Y., Zhao K., Zhu X., Chen Y., Wu B., Zhu F., Zhu B, & Cui L. (2020). A reverse-transcription recombinase-aided amplification assay for the rapid detection of N gene of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). Virology, 549, 1-4.

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Quantification of Microbial Source Tracking Indicators

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In this study, a total of 7 MST indicators were determined, including mtDNA of horse, chicken, cow, human, dog, pig, and sheep. Moreover, 16Sr DNA was determined to calculate the relative abundance of MST indicators. All premiers of target genes are shown in Table S1. Quantification was conducted with a real-time qPCR instrument (ABI QuantStudio^®3, Thermo Fisher Scientific, Waltham, MA, USA). The plasmids containing the MST indicator fragments were constructed with the pUC57 vector (Sangon Biotech, Shanghai, China) and used for the standard curves.
The 20 μL reactions contained 8.2 μL ddH₂O, 0.4 μL forward and reverse primer, 10 μL SYBR Premix Ex TaqTM (Tli RNaseH Plus, TaKaRa, Japan), and 1.0 μL sediment DNA. The following reaction program was run as follows: initial denaturation (2 min at 95 °C), followed by 40 cycles consisting of 10 s at 95 °C, annealing (30 s at annealing temperature described in Table S2), extension (45 s at 72 °C), and a final extension (6 min at 72 °C). Each reaction was run in triplicate for each sample, and sterile water was used as the blank control.

Zhang K., Li K., Liu Z., Li Q., Li W., Chen Q., Xia Y., Hu F, & Yang F. (2022). The Sources and Potential Hosts Identification of Antibiotic Resistance Genes in the Yellow River, Revealed by Metagenomic Analysis. International Journal of Environmental Research and Public Health, 19(16), 10420.

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APOE Genotyping Protocol for Research

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All PCR primers and TaqMan probes were synthesized and purified by Life Technologies (Table 1). Three 197-bp DNA sequences covering the two SNPs for APOE ε2, ε3 or ε4 allele and a 179-bp DNA sequence of beta-actin gene (ACTB) were synthesized and cloned into pUC57 vector, which served as positive control DNA templates (Sangon Biotech, Shanghai, China). All constructs were verified by DNA sequencing (Sangon Biotech, Shanghai, China). Premix PrimeSTAR HS (R040A) was purchased from TAKARA; TaqMan® Genotyping Master Mix (4371357) was purchased from Applied Biosystems; DMSO (D2650) was purchased from Sigma; and blood DNA extraction kit (DP348-03) was purchased from TIANGEN (TIANGEN, Beijing, China).

Zhong L., Xie Y.Z., Cao T.T., Wang Z., Wang T., Li X., Shen R.C., Xu H., Bu G, & Chen X.F. (2016). A rapid and cost-effective method for genotyping apolipoprotein E gene polymorphism. Molecular Neurodegeneration, 11, 2.

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Cloning Alu Sequence into puc57 Vector

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Alu-puc57 vectors were constructed by cloning the entire Alu sequence amplified by PCR into the puc57 vector (Sangon Biotech Co., Ltd., Shanghai, China) at the EcoRI and HindIII restriction sites. The Alu sequence was amplified using a forward primer with an EcoRI restriction site (5′-gaa ttc aga cca tcc tgg cta aca cg-3′) and a reverse primer with a HindIII restriction site (5′-aag ctt aga cgg agt ctc gct ctg tc-3′). PCR was performed at 95°C for 2 min, 94°C for 20 sec, 55°C for 30 sec, and 72°C for 30 sec, repeated for 35 cycles. Extension was performed at 72°C for 10 min and 4°C for 10 min. The reconstructed plasmid, Alu-puc57, was verified by PCR sequencing.

LOU X., HOU Y., LIANG D., PENG L., CHEN H., MA S, & ZHANG L. (2014). A novel Alu-based real-time PCR method for the quantitative detection of plasma circulating cell-free DNA: Sensitivity and specificity for the diagnosis of myocardial infarction. International Journal of Molecular Medicine, 35(1), 72-80.

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Cloning and Expression of CiSA32.6t Gene

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PUC57 vector with the truncated CiSA32.6 (CiSA32.6t) gene (852 bp) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. A set of oligonucleotides, 5′-TAGGATCCGAGAAAAAACTGCAGTTG-3′ and 5′-GCGAATTCTCAAGTAGAGGTAACTTC-3′ (the underlined letters indicate BamH I and EcoR I recognition sites, respectively) and the recombinant plasmid pUC57/CiSA32.6 were used respectively as the primers and the template DNA to amplify the CiSA32.6t by PCR. The purified PCR product and pET28a were doubly digested by BamH I and EcoR I (TAKARA, Dalian, China), respectively. Then, the two products were ligated together and transformed into Escherichia coli BL21.
E. coli containing plasmid pET28a/CiSA32.6t was cultured in liquid LB medium at 37℃ until OD_600nm reached between 0.3 and 0.5. E. coli was induced to express rCiSA32.6t by the addition of isopropylthio-β-D-galactoside (IPTG) (Sangon, Shanghai, China) and incubation for another 6 h at 25℃. rCiSA32.6t was purified by metal affinity chromatography using Ni⁺²-NTA Sepharose (Sangon) according to the instructions from the manufacturer. The purified protein was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis.

Jiang S., Huang X., Li T., Zhang Y, & Zhang J. (2023). Immune response of large yellow croaker Larimichthys crocea towards a recombinant vaccine candidate targeting the parasitic ciliate Cryptocaryon irritans. Aquaculture International.

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Inducible Lentiviral Vector Construction

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The schematic representation of the lentiviral vectors designed in this study was shown in Figure 1. The Tet-on sequence with SnaBI restriction site on the 5’ end and SalI restriction site on the 3’ end was synthesized on pUC57 vector (Sangon Biotech, Shanghai, China). Then the Tet-on sequence was amplified and cloned into SnaBI-SalI digested pCDH-CMV-MCS-EF1α-copGFP vector (System Biosciences, Palo Alto, CA, USA) to create the pCDH-induce vector. To generate the inducible GFP (iGFP) control vector, GFP sequence was amplified from pCDH-CMV-MCS-EF1α-CopGFP vector with primers carrying SalI digest sites, then cloned into SalI digested pCDH-induce vector. To generate iCAR19 transfer vector, the CD19CAR fragment was synthesized with SalI sites at both ends (Sangon Biotech) and ligated into the SalI digested pCDH-induce vector.

Gu X., He D., Li C., Wang H, & Yang G. (2018). Development of Inducible CD19-CAR T Cells with a Tet-On System for Controlled Activity and Enhanced Clinical Safety. International Journal of Molecular Sciences, 19(11), 3455.

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Multiplex Serotyping of E. coli Strains

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There were 3,826 strains stored that were isolated from the stool specimens of infectious diarrheal patients and food samples over 15 years (2003–2018) in Shenzhen Center for Disease Control and Prevention, 3,590 strains were K-serogroups typeable and 236 strains were K-serogroups untypeable (KUT). Among 3,826 strains, 418 strains were previously sequenced (Li et al., 2019 (link); Bian et al., 2021 (link)). Among 418 strains, 338 strains were selected to develop the MLMA assay (Table 1), which included 328 strains representing 39 K-serogroups and 10 strains of untyped K-serogroups for detection of cross-reactions. In addition, 18 rare K-serogroups were incorporated into the assay using Escherichia coli TOP10 strains (n=18, Table S1) containing serogroup-specific genes cloned into a pUC57 vector (Sangon Biotech Co. Ltd., Shanghai, China). Among 3,826 strains, 595 strains which included 10% of 3,590 K-serogroups typeable strains (n=359, Table S2) and all 236 untypeable strains (Table S3) were selected to validate the MLMA assay.

Lu L., Li M., Li Y., Jiang M., Jiang Y., Shi X., Zuo L., Wang L., Bian S., Qiu Y., Cai R., Liao Y., Li Q., Li L, & Hu Q. (2021). A Novel Molecular Method for Simultaneous Identification of Vibrio parahaemolyticus 57 K-Serogroups Using Probe Melting Curve Analysis. Frontiers in Cellular and Infection Microbiology, 11, 594808.

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