LOCUS       Exported                6248 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pING1001, complete sequence.
ACCESSION   KX573874
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6248)
  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 6248)
  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 6248)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6248
                     /organism="Cloning vector pING1001"
                     /mol_type="other DNA"
                     /db_xref="taxon:1881968"
     regulatory      139..167
                     /regulatory_class="promoter"
                     /note="Promoter T7A1"
     protein_bind    144..160
                     /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)."
     protein_bind    177..193
                     /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             240..962
                     /codon_start=1
                     /product="Superfolder GFP"
                     /label=Superfolder GFP
                     /protein_id="AOH95453.1"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYKLE"
     CDS             240..953
                     /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      1013..1064
                     /regulatory_class="terminator"
                     /note="Phage P22 terminator"
     regulatory      1201..1248
                     /regulatory_class="terminator"
                     /note="B. subtilis glnA terminator"
     protein_bind    1260..1278
                     /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      1307..1335
                     /regulatory_class="promoter"
                     /note="Promoter#1"
     CDS             1391..2068
                     /codon_start=1
                     /product="RFP"
                     /label=RFP
                     /protein_id="AOH95454.1"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2113..2184
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     regulatory      2116..2159
                     /regulatory_class="terminator"
                     /note="rrnBT1 terminator"
     terminator      2200..2227
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2362..3036)
                     /codon_start=1
                     /product="REP"
                     /label=REP
                     /protein_id="AOH95456.1"
                     /translation="MEISMATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAA
                     MLLHHLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVS
                     AYVVNDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTG
                     PGEEPPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLP
                     RR"
     CDS             complement(2362..3024)
                     /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      3025..3794
                     /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    3625..5022
                     /label=MOB element
                     /note="MOB element"
     promoter        5172..5263
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5264..6055
                     /codon_start=1
                     /product="AadA1"
                     /label=AadA1
                     /note="aminoglycoside-3-adenyltransferase (Spectinomycin 
                     resistance)"
                     /protein_id="AOH95455.1"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"