pCAP03-acc(3)IV vector (Cat. No.: V007536)

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Basic Information

Note: pCAP03‑acc(3)IV is a yeast‑E. coli‑Streptomyces shuttle vector optimized for high‑efficiency TAR cloning to directly capture large microbial biosynthetic gene clusters in Saccharomyces cerevisiae. It carries TRP1 for positive selection, URA3 for 5‑FOA counter‑selection against NHEJ recircularization, and acc(3)IV for apramycin resistance and convenient linearization, enabling streamlined capture and heterologous expression.

Name:: pCAP03-acc(3)IV

Antibiotic Resistance:: Kanamycin

Length:: 11875 bp

Type:: Bacterial Expression, Yeast Expression

Replication origin:: ori

Selection Marker:: TRP1, URA3 ; apramycin

Copy Number:: High Copy

Promoter:: TRP1

Cloning Method:: Restriction Enzyme

$ 198.8

In stock, 1 week for quality controls

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

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

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

Tang X, Li J, Millán-Aguiñaga N, Zhang JJ, O'Neill EC, Ugalde JA, Jensen PR, Mantovani SM, Moore BS. Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways by Target-directed Genome Mining. ACS Chem Biol. 2015 Dec 18;10(12):2841-2849. doi: 10.1021/acschembio.5b00658. Epub 2015 Oct 21. PMID: 26458099; PMCID: PMC4758359.

pCAP03-acc(3)IV vector (Cat. No.: V007536) Sequence

LOCUS       pCAP03-acc-3-IV        11875 bp    DNA     circular SYN 29-MAY-2026
DEFINITION  pCAP03-acc(3)IV is a upgraded version of the pCAP01(Plasmid #59981).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11875)
  AUTHORS   Tang X, Li J, Millan-Aguinaga N, Zhang JJ, O'Neill EC, Ugalde JA, 
            Jensen PR, Mantovani SM, Moore BS
  TITLE     Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways
            by Target-directed Genome Mining.
  JOURNAL   ACS Chem Biol. 2015 Oct 21.
  PUBMED    26458099
REFERENCE   2  (bases 1 to 11875)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 11875)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 11875)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Chem
            Biol. 2015 Oct 21."
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..11875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          join(7..11875,1..6)
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..704
                     /label=pAdh1  promoter
     protein_bind    739..772
                     /label=FRT (minimal)
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     oriT            complement(779..888)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     CDS             1204..2004
                     /codon_start=1
                     /label=ApmR
                     /note="aminoglycoside 3-N-acetyltransferase type IV"
                     /translation="MSLAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     protein_bind    2014..2060
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2083..2883
                     /codon_start=1
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLATGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     misc_feature    2964..3467
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     primer_bind     3508..3526
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     primer_bind     complement(3564..3586)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3686..3705
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     promoter        3725..4006
                     /label=TRP1 promoter
     CDS             4007..4678
                     /codon_start=1
                     /label=TRP1
                     /note="phosphoribosylanthranilate isomerase, required for 
                     tryptophan biosynthesis"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     primer_bind     complement(4921..4942)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      5189..5777
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5931..5948
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    complement(6162..6296)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6721..6741)
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     intron          complement(6871..6936)
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(7359..8150)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(8937..9305)
                     /codon_start=1
                     /label=traJ
                     /note="oriT-recognizing protein"
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            complement(9338..9447)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     CDS             complement(9685..9696)
                     /codon_start=1
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
     protein_bind    9767..9866
                     /label=phage phi-C31 attP
                     /note="attachment site of phage phi-C31"
     CDS             9876..11690
                     /codon_start=1
                     /label=phage phi-C31 integrase
                     /note="integrase from phage phi-C31"
                     /translation="VDTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDG
                     GRFRFVGHFSEAPGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIP
                     IVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKSAKILDTKNLQREL
                     GGYVGGKAPYGFELVSETKEITRNGRMVNVVINKLAHSTTPLTGPFEFEPDVIRWWWRE
                     IKTHKHLPFKPGSQAAIHPGSITGLCKRMDADAVPTRGETIGKKTASSAWDPATVMRIL
                     RAPRIAGFAAEVIYKKKPDGTPTTKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQA
                     WLDGRGRGKGLSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPG
                     QHEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEAPEKSGERA
                     NLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAELEAAE
                     APKLPLDQWFPEDADADPTGPKSWWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPI
                     EKRASITWAKPPTDDDEDDAQDGTEDVAA"