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  JOURNAL   Unpublished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