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pTpPuc3 vector (V002564#)

Basic Vector Information

Basic Vector Information
Vector Name pTpPuc3 Antibiotic Resistance Kanamycin
Length 7796 bp Type Cloning vector
Source Karas BJ, Diner RE, Lefebvre SC, Weyman PD.

pTpPuc3 vector Vector Map

OriT from RP4 M13 fwd Nourseothricin resistance (Nat) cassette for T. pseudonana yeast Cen6-ArsH4-His3 M13 rev lac operator lac promoter CAP binding site MB1 origin of replication KanR gene from ACYC177 AmpR promoter

Plasmid Resuspension 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 ℃.

pTpPuc3 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                7796 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTpPuc3, complete sequence.
ACCESSION   KP745603
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7796)
  AUTHORS   Karas BJ, Diner RE, Lefebvre SC, Weyman PD.
  TITLE     Designer Diatom episomes Delivered by Bacterial Conjugation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7796)
  AUTHORS   Karas BJ, Diner RE, Lefebvre SC, Weyman PD.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-FEB-2015) Synthetic Biology and Bioenergy Group, J. 
            Craig Venter Institute, 4120 Capricorn Lane, La Jolla, CA 92037, USA
REFERENCE   3  (bases 1 to 7796)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7796
                     /organism="Cloning vector pTpPuc3"
                     /mol_type="other DNA"
                     /label=episome vector
                     /note="episome vector"
                     /db_xref="taxon:1655162"
     misc_feature    185..956
                     /label=OriT from RP4
                     /note="OriT from RP4"
     CDS             complement(307..678)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            711..820
                     /note="incP origin of transfer"
     primer_bind     1098..1114
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1116..3178
                     /note="Nourseothricin resistance (Nat) cassette for T. 
                     pseudonana"
     CDS             2096..2665
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDAGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR
                     LTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGEQALYMSMPCP"
     misc_feature    3179..4561
                     /label=yeast Cen6-ArsH4-His3
                     /note="yeast Cen6-ArsH4-His3"
     misc_feature    3185..3688
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        3689..3876
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             3877..4536
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     primer_bind     complement(4605..4621)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4629..4645
                     /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(4653..4683)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4698..4719
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    complement(5007..5595)
                     /label=MB1 origin of replication
                     /note="MB1 origin of replication"
     rep_origin      complement(5007..5595)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    6320..7289
                     /label=KanR gene from ACYC177
                     /note="KanR gene from ACYC177"
     CDS             6443..7258
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(7597..7701)
                     /gene="bla"
                     /label=AmpR promoter

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