LOCUS       Exported                7506 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pLC290, complete sequence.
ACCESSION   KC296704
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7506)
  AUTHORS   Chubiz LM, Purswani J, Carroll SM, Marx CJ.
  TITLE     A novel pair of inducible expression vectors for use in 
            Methylobacterium extorquens
  JOURNAL   BMC Res Notes 6, 183 (2013)
  PUBMED    23648175
REFERENCE   2  (bases 1 to 7506)
  AUTHORS   Chubiz LM, Purswani J, Marx CJ.
  TITLE     A novel set of broad-host, inducible expression vectors for use in 
            Methylobacterium extorquens and other gram-negative bacteria
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 7506)
  AUTHORS   Chubiz LM, Purswani J, Marx CJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-DEC-2012) Organsmic and Evolutionary Biology, Harvard 
            University, 16 Divinity Ave., Cambridge, MA 02138, USA
REFERENCE   4  (bases 1 to 7506)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7506
                     /organism="Expression vector pLC290"
                     /mol_type="other DNA"
                     /db_xref="taxon:1291080"
     rep_origin      4..712
                     /label=oriV
                     /note="oriV"
     rep_origin      11..642
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      1105..1693
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      1180..1735
                     /label=ColE1
                     /note="ColE1"
     protein_bind    1981..2002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    2011..2053
                     /label=pLac
                     /note="pLac"
     promoter        2017..2047
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2055..2071
                     /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)."
     primer_bind     2079..2095
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2131..2751
                     /codon_start=1
                     /gene="cymR"
                     /product="F1 cumate repressor"
                     /label=cymR
                     /protein_id="AGE65908.1"
                     /translation="MVIMSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPG
                     AAGVSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEF
                     FLDDDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDIL
                     WLIFNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     gene            2131..2751
                     /gene="cymR"
                     /label=cymR
     CDS             2140..2751
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     primer_bind     2794..2810
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      2816..3279
                     /regulatory_class="terminator"
                     /note="TrrnB"
     terminator      2992..3078
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3170..3197
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     misc_feature    3281..3362
                     /label=pR
                     /note="pR"
     protein_bind    3362..3388
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     misc_feature    3363..3388
                     /label=cmtO
                     /note="cmtO"
     CDS             complement(3944..4759)
                     /codon_start=1
                     /gene="kan"
                     /product="aminoglycoside 3-phosphotransferase"
                     /label=kan
                     /protein_id="AGE65907.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(3944..4759)
                     /gene="kan"
                     /label=kan
     CDS             complement(5431..6579)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     misc_feature    6761..7499
                     /label=oriT
                     /note="oriT"
     CDS             complement(6851..7222)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            7255..7364
                     /note="incP origin of transfer"