LOCUS       40924_27588        7506 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pLC290, complete sequence.
ACCESSION   .
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  JOURNAL   Unpublished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)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 7506)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC Res 
            Notes 6, 183 (2013)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (10-DEC-2012) Organsmic and Evolutionary Biology, Harvard 
            University, 16 Divinity Ave., Cambridge, MA 02138, USA"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      3..712
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      1105..1693
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    1981..2002
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2017..2047
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2055..2071
                     /label=lac operator
                     /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             2140..2748
                     /codon_start=1
                     /label=CymR
                     /note="cumate repressor (Mullick et al., 2006)"
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     primer_bind     2794..2810
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      2992..3078
                     /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(3947..4759)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5434..6579)
                     /codon_start=1
                     /label=trfA
                     /note="trans-acting replication protein that binds to and 
                     activates oriV"
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     CDS             complement(6851..7222)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            complement(7255..7364)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
ORIGIN
        1 gaccctttcc gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg
       61 gccctgcaaa cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc
      121 gttgtggata cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca
      181 cttgaggggc cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc
      241 cggcgacgtg gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt
      301 tcccacagat gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg
      361 gcgcgactac tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca
      421 gatgaggggc gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt
      481 gcaagggttt ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac
      541 caatatttat aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc
      601 cgaagggggg tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc
      661 ccccaggggc tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agccaagctg
      721 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
      781 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
      841 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
      901 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
      961 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     1021 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1081 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     1141 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1201 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     1261 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1321 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1381 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1441 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     1501 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     1561 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     1621 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     1681 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     1741 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     1801 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     1861 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     1921 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     1981 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     2041 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     2101 ttacgccaag ctagcaggga gagaccccga atggtgatca tgagtccaaa gagaagaaca
     2161 caggcagagc gcgcaatgga gacccagggc aagttgattg cagcggccct gggggtttta
     2221 cgggaaaaag gttacgcggg attccggatc gcagatgtgc ccggtgctgc aggtgtctcg
     2281 agaggagcgc agagccatca tttcccgaca aagcttgagc ttctgcttgc cacttttgaa
     2341 tggctttacg aacagatcac cgaacgcagt cgggctcgat tagcgaaatt gaagccagag
     2401 gatgacgtca tccagcaaat gctggacgac gccgccgaat ttttcctcga cgatgacttc
     2461 tctatcagcc ttgatttgat tgtggctgcc gaccgggatc cagcgttacg cgagggtatt
     2521 cagcgcacgg tagagaggaa tcggtttgtc gtcgaggata tgtggcttgg tgttctggtg
     2581 agccgtggtc tttcgcgcga tgatgcagaa gatatccttt ggttgatatt caattcggtg
     2641 cgtgggcttg ctgttcgtag cctatggcag aaggacaaag aacgctttga gcgtgtcagg
     2701 aactcgacac tcgaaattgc gcgagagcgg tacgcgaaat tcaagcgcta gggccaagtt
     2761 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaatttttca
     2821 gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg
     2881 gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg
     2941 ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa
     3001 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     3061 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     3121 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc
     3181 ctgacggatg gcctttttgc gtttctacaa actcttcctg tcgtcatatc tacaagccat
     3241 ccccccacag atacggtaaa ctagcctcgt ttttgcatcc caacaactta taccatggcc
     3301 tacaaaaagg caaacaatgg tacttgacga ctcatcacaa caattgtagt tgtagattgt
     3361 aaacaaacag acaatctggt ctgtttgtgg tacctctaga agatctacgc gtactagtgc
     3421 atgcgagctc accggtgaat tcacgtgcgc acctgtgctg ggcgcgctgt cggatcgttt
     3481 cgggcggcgg ccaatcttgc tcgtctcgct ggccggcgcc actgtcgact acgccatcat
     3541 ggcgacagcg cctttccttt gggttctcta tatcgggcgg atcgtggccg gcatcaccgg
     3601 ggcgactggg gcggtagccg gcgcttatat tgccgatgac ctgcaggggg gggggggcgc
     3661 tgaggtctgc ctcgtgaaga aggtgttgct gactcatacc aggcctgaat cgccccatca
     3721 tccagccaga aagtgaggga gccacggttg atgagagctt tgttgtaggt ggaccagttg
     3781 gtgattttga acttttgctt tgccacggaa cggtctgcgt tgtcgggaag atgcgtgatc
     3841 tgatccttca actcagcaaa agttcgattt attcaacaaa gccgccgtcc cgtcaagtca
     3901 gcgtaatgct ctgccagtgt tacaaccaat taaccaattc tgattagaaa aactcatcga
     3961 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     4021 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     4081 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     4141 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     4201 gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     4261 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     4321 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     4381 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     4441 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     4501 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     4561 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     4621 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     4681 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgag caagacgttt
     4741 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     4801 ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa
     4861 cgtggctttc cccccccccc ctgcaggtcc gacacgggga tggatggcgt tcccgatcat
     4921 ggtcctgctt gcttcgggtg gcatcggaat gccggcgctg caagcaatgt tgtccaggca
     4981 ggtggatgag gaacgtcagg ggcagctgca aggctcactg gcggcgctca ccagcctgac
     5041 ctcgatcgtc ggacccctcc tcttcacggc gatctatgcg gcttctataa caacgtggaa
     5101 cgggtgggca tggattgcag gcgctgccct ctacttgctc tgcctgccgg cgctgcgtcg
     5161 cgggctttgg agcggcgcag ggcaacgagc cgatcgctga tcgtggaaac gataggccta
     5221 tgccatgcgg gtcaaggcga cttccggcaa gctatacgcg ccctagaatt gtcaatttta
     5281 atcctctgtt tatcggcagt tcgtagagcg cgccgtgcgt cccgagcgat actgagcgaa
     5341 gcaagtgcgt cgagcagtgc ccgcttgttc ctgaaatgcc agtaaagcgc tggctgctga
     5401 acccccagcc ggaactgacc ccacaaggcc ctagcgtttg caatgcacca ggtcatcatt
     5461 gacccaggcg tgttccacca ggccgctgcc tcgcaactct tcgcaggctt cgccgacctg
     5521 ctcgcgccac ttcttcacgc gggtggaatc cgatccgcac atgaggcgga aggtttccag
     5581 cttgagcggg tacggctccc ggtgcgagct gaaatagtcg aacatccgtc gggccgtcgg
     5641 cgacagcttg cggtacttct cccatatgaa tttcgtgtag tggtcgccag caaacagcac
     5701 gacgatttcc tcgtcgatca ggacctggca acgggacgtt ttcttgccac ggtccaggac
     5761 gcggaagcgg tgcagcagcg acaccgattc caggtgccca acgcggtcgg acgtgaagcc
     5821 catcgccgtc gcctgtaggc gcgacaggca ttcctcggcc ttcgtgtaat accggccatt
     5881 gatcgaccag cccaggtcct ggcaaagctc gtagaacgtg aaggtgatcg gctcgccgat
     5941 aggggtgcgc ttcgcgtact ccaacacctg ctgccacacc agttcgtcat cgtcggcccg
     6001 cagctcgacg ccggtgtagg tgatcttcac gtccttgttg acgtggaaaa tgaccttgtt
     6061 ttgcagcgcc tcgcgcggga ttttcttgtt gcgcgtggtg aacagggcag agcgggccgt
     6121 gtcgtttggc atcgctcgca tcgtgtccgg ccacggcgca atatcgaaca aggaaagctg
     6181 catttccttg atctgctgct tcgtgtgttt cagcaacgcg gcctgcttgg cctcgctgac
     6241 ctgttttgcc aggtcctcgc cggcggtttt tcgcttcttg gtcgtcatag ttcctcgcgt
     6301 gtcgatggtc atcgacttcg ccaaacctgc cgcctcctgt tcgagacgac gcgaacgctc
     6361 cacggcggcc gatggcgcgg gcagggcagg gggagccagt tgcacgctgt cgcgctcgat
     6421 cttggccgta gcttgctgga ccatcgagcc gacggactgg aaggtttcgc ggggcgcacg
     6481 catgacggtg cggcttgcga tggtttcggc atcctcggcg gaaaaccccg cgtcgatcag
     6541 ttcttgcctg tatgccttcc ggtcaaacgt ccgattcatt caccctcctt gcgggattgc
     6601 cccgactcac gccggggcaa tgtgccctta ttcctgattt gacccgcctg gtgccttggt
     6661 gtccagataa tccaccttat cggcaatgaa gtcggtcccg tagaccgtct ggccgtcctt
     6721 ctcgtacttg gtattccgaa tcttgccctg cacgaatacc agctccgcga agtcgctctt
     6781 cttgatggag cgcatgggga cgtgcttggc aatcacgcgc accccccggc cgttttagcg
     6841 gctaaaaaag tcatggctct gccctcgggc ggaccacgcc catcatgacc ttgccaagct
     6901 cgtcctgctt ctcttcgatc ttcgccagca gggcgaggat cgtggcatca ccgaaccgcg
     6961 ccgtgcgcgg gtcgtcggtg agccagagtt tcagcaggcc gcccaggcgg cccaggtcgc
     7021 cattgatgcg ggccagctcg cggacgtgct catagtccac gacgcccgtg attttgtagc
     7081 cctggccgac ggccagcagg taggcctaca ggctcatgcc ggccgccgcc gccttttcct
     7141 caatcgctct tcgttcgtct ggaaggcagt acaccttgat aggtgggctg cccttcctgg
     7201 ttggcttggt ttcatcagcc atccgcttgc cctcatctgt tacgccggcg gtagccggcc
     7261 agcctcgcag agcaggattc ccgttgagca ccgccaggtg cgaataaggg acagtgaaga
     7321 aggaacaccc gctcgcgggt gggcctactt cacctatcct gcccggctga cgccgttgga
     7381 tacaccaagg aaagtctaca cgaacccttt ggcaaaatcc tgtatatcgt gcgaaaaagg
     7441 atggatatac cgaaaaaatc gctataatga ccccgaagca gggttatgca gcggaaaaga
     7501 tccgtc
//
