LOCUS       40924_29591        3885 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pM13, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3885)
  AUTHORS   Zobel S, Benedetti I, Eisenbach L, de Lorenzo V, Wierckx N, Blank 
            LM.
  TITLE     A Tn7-based device for calibrated heterologous gene expression in 
            Pseudomonas putida
  JOURNAL   ACS Synth Biol (2015) In press
  PUBMED    26133359
REFERENCE   2  (bases 1 to 3885)
  AUTHORS   Zobel S, Benedetti I, Eisenbach L, de Lorenzo V, Wierckx N, Blank 
            LM.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-JUN-2015) System Biology, Centro Nacional de 
            Biotecnologia, C/Darwin 3, Madrid 28049, Spain
REFERENCE   3  (bases 1 to 3885)
  AUTHORS   Zobel S, Benedetti I, Eisenbach L, de Lorenzo V, Wierckx N, Blank 
            LM.
  TITLE     Direct Submission
  JOURNAL   Submitted (31-AUG-2015) System Biology, Centro Nacional de 
            Biotecnologia, C/Darwin 3, Madrid 28049, Spain
REFERENCE   4  (bases 1 to 3885)
  AUTHORS   Zobel S, Benedetti I, Eisenbach L, de Lorenzo V, Wierckx N, Blank 
            LM.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-NOV-2015) System Biology, Centro Nacional de 
            Biotecnologia, C/Darwin 3, Madrid 28049, Spain
REFERENCE   5  (bases 1 to 3885)
  TITLE     Direct Submission
REFERENCE   6  (bases 1 to 3885)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol (2015) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (09-JUN-2015) System Biology, Centro Nacional de Biotecnologia, 
            C/Darwin 3, Madrid 28049, Spain"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (31-AUG-2015) System Biology, Centro Nacional de Biotecnologia, 
            C/Darwin 3, Madrid 28049, Spain"
COMMENT     SGRef: number: 4; type: "Journal Article"; journalName: "Submitted 
            (13-NOV-2015) System Biology, Centro Nacional de Biotecnologia, 
            C/Darwin 3, Madrid 28049, Spain"
COMMENT     SGRef: number: 5; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
            On Nov 13, 2015 this sequence version replaced KT192135.2.
FEATURES             Location/Qualifiers
     source          1..3885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        11..58
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     misc_feature    95..151
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     CDS             166..879
                     /codon_start=1
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Pedelacq et al., 2006)"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      893..987
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     misc_feature    992..1190
                     /label=Tn7R transposase recognition site
                     /note="Tn7R transposase recognition site"
     CDS             1300..2112
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     oriT            2271..2379
                     /label=oriT
                     /note="incP origin of transfer"
     rep_origin      2398..2786
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     mobile_element  2798..2963
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     terminator      complement(2977..3063)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(3132..3662)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(3851..3879)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 ttaattaatt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa
       61 ggtgaggaac taaaccccta ggccgcggcc gcgcgaattc gagctcggta cccggggatc
      121 ctctagagtc gacctgcagg catgcaagct taggaggaaa aacatatgcg taaaggtgaa
      181 gaactgttca ccggtgttgt tccgatcctg gttgaactgg atggtgatgt taacggccac
      241 aaattctctg ttcgtggtga aggtgaaggt gatgcaacca acggtaaact gaccctgaaa
      301 ttcatctgca ctaccggtaa actgccggtt ccatggccga ctctggtgac taccctgacc
      361 tatggtgttc agtgtttttc tcgttacccg gatcacatga agcagcatga tttcttcaaa
      421 tctgcaatgc cggaaggtta tgtacaggag cgcaccattt ctttcaaaga cgatggcacc
      481 tacaaaaccc gtgcagaggt taaatttgaa ggtgatactc tggtgaaccg tattgaactg
      541 aaaggcattg atttcaaaga ggacggcaac atcctgggcc acaaactgga atataacttc
      601 aactcccata acgtttacat caccgcagac aaacagaaga acggtatcaa agctaacttc
      661 aaaattcgcc ataacgttga agacggtagc gtacagctgg cggaccacta ccagcagaac
      721 actccgatcg gtgatggtcc ggttctgctg ccggataacc actacctgtc cacccagtct
      781 aaactgtcca aagacccgaa cgaaaagcgc gaccacatgg tgctgctgga gttcgttact
      841 gccgcaggta tcacgcacgg catggatgaa ctctacaaat aaactagtct tggactcctg
      901 ttgatagatc cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc
      961 cgccgggcgt tttttattgg tgagaatcca gccacgcccc tctttaatac gacgggcaat
     1021 ttgcacttca gaaaatgaag agtttgcttt agccataaca aaagtccagt atgctttttc
     1081 acagcataac tggactgatt tcagtttaca actattctgt ctagtttaag actttattgt
     1141 catagtttag atctattttg ttcagtttaa gactttattg tccgcccaca atttaaattt
     1201 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     1261 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcagcgtgaa
     1321 acgagctgta gccgtccgcg tctgaacagc aacatggatg cggatctgta tggctataaa
     1381 tgggcgcgtg ataacgtggg tcagagcggc gcgaccattt atcgtctgta tggcaaaccg
     1441 gatgcgccgg aactgtttct gaaacatggc aaaggcagcg tggcgaacga tgtgaccgat
     1501 gaaatggtgc gtctgaactg gctgaccgaa tttatgccgc tgccgaccat taaacatttt
     1561 attcgcaccc cggatgatgc gtggctgctg accaccgcga ttccgggcaa aaccgcgttt
     1621 caggtgctgg aagaatatcc ggatagcggc gaaaacattg tggatgcgct ggccgtgttt
     1681 ctgcgtcgtc tgcatagcat tccggtgtgc aactgcccgt ttaacagcga tcgtgtgttt
     1741 cgtctggccc aggcgcagag ccgtatgaac aacggcctgg tggatgcgag cgattttgat
     1801 gatgaacgta acggctggcc ggtggaacag gtgtggaaag aaatgcataa actgctgccg
     1861 tttagcccgg atagcgtggt gacccacggc gattttagcc tggataacct gattttcgat
     1921 gaaggcaaac tgattggctg cattgatgtg ggccgtgtgg gcattgcgga tcgttatcag
     1981 gatctggcca ttctgtggaa ctgcctgggc gaatttagcc cgagcctgca aaaacgtctg
     2041 tttcagaaat atggcattga taatccggat atgaacaaac tgcaatttca tctgatgctg
     2101 gatgaatttt tctaataatt aattggaccg cggtcctttt ccgctgcata accctgcttc
     2161 ggggtcatta tagcgatttt ttcggtatat ccatcctttt tcgcacgata tacaggattt
     2221 tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc gggcaggata
     2281 ggtgaagtag gcccacccgc gagcgggtgt tccttcttca ctgtccctta ttcgcacctg
     2341 gcggtgctca acgggaatcc tgctctgcga ggctggccgt aggccggccg gcgcgccgat
     2401 ctgaagatca gcagttcaac ctgttgatag tacgtactaa gctctcatgt ttcacgtact
     2461 aagctctcat gtttaacgta ctaagctctc atgtttaacg aactaaaccc tcatggctaa
     2521 cgtactaagc tctcatggct aacgtactaa gctctcatgt ttcacgtact aagctctcat
     2581 gtttgaacaa taaaattaat ataaatcagc aacttaaata gcctctaagg ttttaagttt
     2641 tataagaaaa aaaagaatat ataaggcttt taaagccttt aaggtttaac ggttgtggac
     2701 aacaagccag ggatgtaacg cactgagaag cccttagagc ctctcaaagc aattttgagt
     2761 gacacaggaa cacttaacgg ctgacatggg gcgcgcctgt gggcggacaa aatagttggg
     2821 aactgggagg ggtggaaatg gagtttttaa ggattattta gggaagagtg acaaaataga
     2881 tgggaactgg gtgtagcgtc gtaagctaat acgaaaatta aaaatgacaa aatagtttgg
     2941 aactagattt cacttatctg gtttctaggg cggcggattt gtcctactca ggagagcgtt
     3001 caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat
     3061 ttgatgccga cgtccaattt accgaacaac tccgcggccg ggaagccgat ctcggcttga
     3121 acgaattgtt aggtggcggt acttgggtcg atatcaaagt gcatcacttc ttcccgtatg
     3181 cccaactttg tatagagagc cactgcggga tcgtcaccgt aatctgcttg cacgtagatc
     3241 acataagcac caagcgcgtt ggcctcatgc ttgaggagat tgatgagcgc ggtggcaatg
     3301 ccctgcctcc ggtgctctcc ggagactgcg agatcataga tatagatctc actacgcggc
     3361 tgctcaaact tgggcagaac gtaagccgcg agagcgccaa caaccgcttc ttggtcgaag
     3421 gcagcaagcg cgatgaatgt cttactacgg agcaagttcc cgaggtaatc ggagtccggc
     3481 tgatgttggg agtaggtggc tacgtctccg aactcacgac cgaaaagatc aagagcagcc
     3541 cgcatggatt tgacttggtc agggccgagc ctacatgtgc gaatgatgcc catacttgag
     3601 ccacctaact ttgttttagg gcgactgccc tgctgcgtaa catcgttgct gctgcgtaac
     3661 atcgttgctg ctccataaca tcaaacatcg acccacggcg taacgcgctt gctgcttgga
     3721 tgcccgaggc ataggctgta caaaaaaaca gtcataacaa gccatgaaaa ccgccactgc
     3781 gccgttacca ccgctgcgtt cggtcaaggt tctggaccag ttgcgtgagc gcatacgcta
     3841 cttgcattac agtttacgaa ccgaacaggc ttatgtcaag acgtc
//
