LOCUS       Exported                7140 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPA1, complete sequence.
ACCESSION   KX573876
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7140)
  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 7140)
  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 7140)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7140
                     /organism="Cloning vector pPA1"
                     /mol_type="other DNA"
                     /db_xref="taxon:1881969"
     CDS             complement(158..820)
                     /codon_start=1
                     /product="REP"
                     /label=REP
                     /protein_id="AOH95461.1"
                     /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    complement(1421..2818)
                     /label=Mob element
                     /note="Mob element"
     promoter        2968..3059
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3060..3851
                     /codon_start=1
                     /product="AdhA1"
                     /label=AdhA1
                     /note="aminoglycoside-3-adenyltransferase"
                     /protein_id="AOH95462.1"
                     /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#27"
     regulatory      4236..4244
                     /regulatory_class="ribosome_binding_site"
                     /note="pdc RBS#3"
     CDS             4250..5956
                     /codon_start=1
                     /product="Pdc"
                     /label=Pdc
                     /note="Pyruvate Decarboxylase (Codon optimized)"
                     /protein_id="AOH95463.1"
                     /translation="MSYTVGTYLAERLVQIGLKHHFAVAGDYNLVLLDNLLLNKNMEQV
                     YCCNELNCGFSAEGYARAKGAAAAVVTYSVGALSAFDAIGGAYAENLPVILISGAPNNN
                     DHAAGHVLHHALGKTDYHYQLEMAKNITAAAEAIYTPEEAPAKIDHVIKTALREKKPVY
                     LEIACNIASMPCAAPGPASALFNDEASDEASLNAAVEETLKFIANRDKVAVLVGSKLRA
                     AGAEEAAVKFADALGGAVATMAAAKSFFPEENPHYIGTSWGEVSYPGVEKTMKEADAVI
                     ALAPVFNDYSTTGWTDIPDPKKLVLAEPRSVVVNGVRFPSVHLKDYLTRLAQKVSKKTG
                     ALDFFKSLNAGELKKAAPADPSAPLVNAEIARQVEALLTPNTTVIAETGDSWFNAQRMK
                     LPNGARVEYEMQWGHIGWSVPAAFGYAVGAPERRNILMVGDGSFQLTAQEVAQMVRLKL
                     PVIIFLINNYGYTIEVMIHDGPYNNIKNWDYAGLMEVFNGNGGYDSGAGKGLKAKTGGE
                     LAEAIKVALANTDGPTLIECFIGREDCTEELVKWGKRVAAANSRKPVNKLL"
     regulatory      5972..5980
                     /regulatory_class="ribosome_binding_site"
                     /note="adhA RBS#2"
     CDS             5986..6999
                     /codon_start=1
                     /product="AdhA"
                     /label=AdhA
                     /note="Alcohol Dehydrogenase I (Codon optimized)"
                     /protein_id="AOH95464.1"
                     /translation="MKAAVITKDHTIEVKDTKLRPLKYGEALLEMEYCGVCHTDLHVKN
                     GDFGDETGRITGHEGIGIVKQVGEGVTSLKVGDRASVAWFFKGCGHCEYCVSGNETLCR
                     NVENAGYTVDGAMAEECIVVADYSVKVPDGLDPAVASSITCAGVTTYKAVKVSQIQPGQ
                     WLAIYGLGGLGNLALQYAKNVFNAKVIAIDVNDEQLAFAKELGADMVINPKNEDAAKII
                     QEKVGGAHATVVTAVAKSAFNSAVEAIRAGGRVVAVGLPPEKMDLSIPRLVLDGIEVLG
                     SLVGTREDLKEAFQFAAEGKVKPKVTKRKVEEINQIFDEMEHGKFTGRMVVDFTHH"
     regulatory      7044..7095
                     /regulatory_class="terminator"
                     /note="Phage P22 terminator"