LOCUS       Exported                7284 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPB1, complete sequence.
ACCESSION   KX573875
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7284)
  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 7284)
  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 7284)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7284
                     /organism="Cloning vector pPB1"
                     /mol_type="other DNA"
                     /db_xref="taxon:1881970"
     CDS             complement(158..832)
                     /codon_start=1
                     /product="REP"
                     /label=REP
                     /protein_id="AOH95457.1"
                     /translation="MEISMATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAA
                     MLLHHLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVS
                     AYVVNDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTG
                     PGEEPPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLP
                     RR"
     CDS             complement(158..820)
                     /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      821..1590
                     /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    1421..2818
                     /label=Mob element
                     /note="Mob element"
     promoter        2968..3059
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3060..3851
                     /codon_start=1
                     /product="AadA1"
                     /label=AadA1
                     /note="aminoglycoside-3-adenyltransferase (Spectinomycin 
                     resistance)"
                     /protein_id="AOH95458.1"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     regulatory      4048..4095
                     /regulatory_class="terminator"
                     /note="B. subtilis glnA terminator"
     protein_bind    4107..4125
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     regulatory      4154..4182
                     /regulatory_class="promoter"
                     /note="Promoter#25"
     CDS             4181..5956
                     /codon_start=1
                     /product="Pdc"
                     /label=Pdc
                     /note="Pyruvate Decarboxylase (Codon optimized)"
                     /protein_id="AOH95459.1"
                     /translation="MCGIGRVFLLRGHLKSTILRRCYMSYTVGTYLAERLVQIGLKHHF
                     AVAGDYNLVLLDNLLLNKNMEQVYCCNELNCGFSAEGYARAKGAAAAVVTYSVGALSAF
                     DAIGGAYAENLPVILISGAPNNNDHAAGHVLHHALGKTDYHYQLEMAKNITAAAEAIYT
                     PEEAPAKIDHVIKTALREKKPVYLEIACNIASMPCAAPGPASALFNDEASDEASLNAAV
                     EETLKFIANRDKVAVLVGSKLRAAGAEEAAVKFADALGGAVATMAAAKSFFPEENPHYI
                     GTSWGEVSYPGVEKTMKEADAVIALAPVFNDYSTTGWTDIPDPKKLVLAEPRSVVVNGV
                     RFPSVHLKDYLTRLAQKVSKKTGALDFFKSLNAGELKKAAPADPSAPLVNAEIARQVEA
                     LLTPNTTVIAETGDSWFNAQRMKLPNGARVEYEMQWGHIGWSVPAAFGYAVGAPERRNI
                     LMVGDGSFQLTAQEVAQMVRLKLPVIIFLINNYGYTIEVMIHDGPYNNIKNWDYAGLME
                     VFNGNGGYDSGAGKGLKAKTGGELAEAIKVALANTDGPTLIECFIGREDCTEELVKWGK
                     RVAAANSRKPVNKLL"
     regulatory      4236..4244
                     /regulatory_class="ribosome_binding_site"
                     /note="pdc RBS#3"
     CDS             5972..7144
                     /codon_start=1
                     /product="AdhB"
                     /label=AdhB
                     /note="Alcohol Dehydrogenase II (Codon optimized)"
                     /protein_id="AOH95460.1"
                     /translation="MALGGIAMASSTFYIPFVNEMGEGSLEKAIKDLNGSGFKNALIVS
                     DAFMNKSGVVKQVADLLKAQGINSAVYDGVMPNPTVTAVLEGLKILKDNNSDFVISLGG
                     GSPHDCAKAIALVATNGGEVKDYEGIDKSKKPALPLMSINTTAGTASEMTRFCIITDEV
                     RHVKMAIVDRHVTPMVSVNDPLLMVGMPKGLTAATGMDALTHAFEAYSSTAATPITDAC
                     ALKAASMIAKNLKTACDNGKDMPAREAMAYAQFLAGMAFNNASLGYVHAMAHQLGGYYN
                     LPHGVCNAVLLPHVLAYNASVVAGRLKDVGVAMGLDIANLGDKEGAEATIQAVRDLAAS
                     IGIPANLTELGAKKEDVPLLADHALKDACALTNPRQGDQKEVEELFLSAF"
     regulatory      5979..5987
                     /regulatory_class="ribosome_binding_site"
                     /note="adhB RBS#1"
     regulatory      7188..7239
                     /regulatory_class="terminator"
                     /note="Phage P22 terminator"