LOCUS       Exported                6389 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRH52, complete sequence.
ACCESSION   KX573872
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6389)
  AUTHORS   Ghosh IN, Landick R.
  TITLE     OptSSeq: High-throughput sequencing readout of growth enrichment 
            defines optimal gene expression elements for homoethanologenesis
  JOURNAL   ACS Synth Biol (2016) In press
  PUBMED    27404024
REFERENCE   2  (bases 1 to 6389)
  AUTHORS   Ghosh IN, Landick RC.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-JUL-2016) DOE Great Lakes Bioenergy Research Center, 
            University of Wisconsin-Madison, 1552 University Avenue, Madison, WI
            53726, United States
REFERENCE   3  (bases 1 to 6389)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     Cloning vector recieved with thanks from Haft, R.J.
FEATURES             Location/Qualifiers
     source          1..6389
                     /organism="Cloning vector pRH52"
                     /mol_type="other DNA"
                     /db_xref="taxon:1881974"
     promoter        163..240
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     regulatory      177..205
                     /regulatory_class="promoter"
                     /note="LacI promoter"
     CDS             364..1323
                     /codon_start=1
                     /product="LacI"
                     /label=LacI
                     /note="Lac repressor protein"
                     /protein_id="AOH95446.1"
                     /translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSR
                     ADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPA
                     LFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKY
                     LTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLR
                     VGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVS
                     LVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ"
     regulatory      1326..1373
                     /regulatory_class="terminator"
                     /note="B. subtilis glnA terminator"
     protein_bind    1385..1404
                     /allele="lacOid"
                     /label=LacI binding site
                     /bound_moiety="LacI"
     protein_bind    1404..1425
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     regulatory      1440..1468
                     /regulatory_class="promoter"
                     /note="Trc promoter (Ptrc); Trc promoter (Ptrc)"
     protein_bind    1475..1495
                     /allele="lacO1"
                     /label=LacI binding site
                     /bound_moiety="LacI"
     protein_bind    1477..1493
                     /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)."
     CDS             1518..1568
                     /codon_start=1
                     /product="LazZ alpha"
                     /label=LazZ alpha
                     /protein_id="AOH95450.1"
                     /translation="MAMITPSAQLTLTKGN"
     promoter        1543..1561
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     RBS             1597..1619
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1627..2349
                     /codon_start=1
                     /product="Superfolder GFP"
                     /label=Superfolder GFP
                     /protein_id="AOH95447.1"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYKLE"
     CDS             1627..2340
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     regulatory      2400..2451
                     /regulatory_class="terminator"
                     /note="Phage 22 terminator"
     CDS             complement(2654..3328)
                     /codon_start=1
                     /product="REP"
                     /label=REP
                     /protein_id="AOH95448.1"
                     /translation="MEISMATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAA
                     MLLHHLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVS
                     AYVVNDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTG
                     PGEEPPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLP
                     RR"
     CDS             complement(2654..3316)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      3317..4086
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     misc_feature    3917..5314
                     /label=MOB element
                     /note="MOB element"
     promoter        5464..5555
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5556..6347
                     /codon_start=1
                     /product="AadA1"
                     /label=AadA1
                     /note="Aminoglycoside-3-adenylyltransferase (Spectinomycon 
                     resistance)"
                     /protein_id="AOH95449.1"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"