LOCUS       40924_25641        3846 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector PIR2-m1, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3846)
  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 3846)
  AUTHORS   Zobel S, Benedetti I, Eisenbach L, de Lorenzo V, Wierckx N, Blank 
            LM.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-JUN-2015) System Biology, Centro Nacional de 
            Biotecnologia, C/Darwin 3, Madrid 28049, Spain
REFERENCE   3  (bases 1 to 3846)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 3846)
  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 
            (15-JUN-2015) System Biology, Centro Nacional de Biotecnologia, 
            C/Darwin 3, Madrid 28049, Spain"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..3846
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    56..112
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     CDS             127..840
                     /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      854..948
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     misc_feature    953..1151
                     /label=Tn7R transposase recognition site
                     /note="Tn7R transposase recognition site"
     CDS             1280..2032
                     /codon_start=1
                     /gene="aphA"
                     /product="aminoglycoside 3'-phosphotransferase"
                     /label=aphA
                     /note="kanamycin resistance marker"
                     /protein_id="AKQ71012.1"
                     /translation="MLYGYKWARDNVGQSGATIYRLYGKPDAPELFLKHGKGSVANDVT
                     DEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAFQVLEEYPDSGENIVDALA
                     VFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHK
                     LLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSL
                     QKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            1280..2032
                     /gene="aphA"
                     /label=aphA
                     /note="aminoglycoside 3'-phosphotransferase"
     oriT            2232..2340
                     /label=oriT
                     /note="incP origin of transfer"
     rep_origin      2359..2747
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     mobile_element  2759..2924
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     terminator      complement(2938..3024)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(3093..3623)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(3812..3840)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 ttaattaaag cggataacaa tttcacacag gaggccgcct aggccgcggc cgcgcgaatt
       61 cgagctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc ttaggaggaa
      121 aaacatatgc gtaaaggtga agaactgttc accggtgttg ttccgatcct ggttgaactg
      181 gatggtgatg ttaacggcca caaattctct gttcgtggtg aaggtgaagg tgatgcaacc
      241 aacggtaaac tgaccctgaa attcatctgc actaccggta aactgccggt tccatggccg
      301 actctggtga ctaccctgac ctatggtgtt cagtgttttt ctcgttaccc ggatcacatg
      361 aagcagcatg atttcttcaa atctgcaatg ccggaaggtt atgtacagga gcgcaccatt
      421 tctttcaaag acgatggcac ctacaaaacc cgtgcagagg ttaaatttga aggtgatact
      481 ctggtgaacc gtattgaact gaaaggcatt gatttcaaag aggacggcaa catcctgggc
      541 cacaaactgg aatataactt caactcccat aacgtttaca tcaccgcaga caaacagaag
      601 aacggtatca aagctaactt caaaattcgc cataacgttg aagacggtag cgtacagctg
      661 gcggaccact accagcagaa cactccgatc ggtgatggtc cggttctgct gccggataac
      721 cactacctgt ccacccagtc taaactgtcc aaagacccga acgaaaagcg cgaccacatg
      781 gtgctgctgg agttcgttac tgccgcaggt atcacgcacg gcatggatga actctacaaa
      841 taaactagtc ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggat
      901 ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc agccacgccc
      961 ctctttaata cgacgggcaa tttgcacttc agaaaatgaa gagtttgctt tagccataac
     1021 aaaagtccag tatgcttttt cacagcataa ctggactgat ttcagtttac aactattctg
     1081 tctagtttaa gactttattg tcatagttta gatctatttt gttcagttta agactttatt
     1141 gtccgcccac aatttaaatt tgacataaga cgtacctgtt cggttcgtaa ttagggactg
     1201 taatgcaagt agcgtatgag ccatattcag cgtgaaacga gctgtagccg tccgcgtctg
     1261 aacagcaaca tggatgcgga tgctgtatgg ctataaatgg gcgcgtgata acgtgggtca
     1321 gagcggcgcg accatttatc gtctgtatgg caaaccggat gcgccggaac tgtttctgaa
     1381 acatggcaaa ggcagcgtgg cgaacgatgt gaccgatgaa atggtgcgtc tgaactggct
     1441 gaccgaattt atgccgctgc cgaccattaa acattttatt cgcaccccgg atgatgcgtg
     1501 gctgctgacc accgcgattc cgggcaaaac cgcgtttcag gtgctggaag aatatccgga
     1561 tagcggcgaa aacattgtgg atgcgctggc cgtgtttctg cgtcgtctgc atagcattcc
     1621 ggtgtgcaac tgcccgttta acagcgatcg tgtgtttcgt ctggcccagg cgcagagccg
     1681 tatgaacaac ggcctggtgg atgcgagcga ttttgatgat gaacgtaacg gctggccggt
     1741 ggaacaggtg tggaaagaaa tgcataaact gctgccgttt agcccggata gcgtggtgac
     1801 ccacggcgat tttagcctgg ataacctgat tttcgatgaa ggcaaactga ttggctgcat
     1861 tgatgtgggc cgtgtgggca ttgcggatcg ttatcaggat ctggccattc tgtggaactg
     1921 cctgggcgaa tttagcccga gcctgcaaaa acgtctgttt cagaaatatg gcattgataa
     1981 tccggatatg aacaaactgc aatttcatct gatgctggat gaatttttct aacaattcgt
     2041 tcaagccgag atcggcttcc cggccgcgga gttgttcggt aaattggaca agggtccttt
     2101 tccgctgcat aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt
     2161 ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa
     2221 cggcgtcagc cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc
     2281 actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg
     2341 taggccggcc ggcgcgccga tctgaagatc agcagttcaa cctgttgata gtacgtacta
     2401 agctctcatg tttcacgtac taagctctca tgtttaacgt actaagctct catgtttaac
     2461 gaactaaacc ctcatggcta acgtactaag ctctcatggc taacgtacta agctctcatg
     2521 tttcacgtac taagctctca tgtttgaaca ataaaattaa tataaatcag caacttaaat
     2581 agcctctaag gttttaagtt ttataagaaa aaaaagaata tataaggctt ttaaagcctt
     2641 taaggtttaa cggttgtgga caacaagcca gggatgtaac gcactgagaa gcccttagag
     2701 cctctcaaag caattttgag tgacacagga acacttaacg gctgacatgg ggcgcgcctg
     2761 tgggcggaca aaatagttgg gaactgggag gggtggaaat ggagttttta aggattattt
     2821 agggaagagt gacaaaatag atgggaactg ggtgtagcgt cgtaagctaa tacgaaaatt
     2881 aaaaatgaca aaatagtttg gaactagatt tcacttatct ggtttctagg gcggcggatt
     2941 tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttt
     3001 cgactgagcc tttcgtttta tttgatgccg acgtccaatt taccgaacaa ctccgcggcc
     3061 gggaagccga tctcggcttg aacgaattgt taggtggcgg tacttgggtc gatatcaaag
     3121 tgcatcactt cttcccgtat gcccaacttt gtatagagag ccactgcggg atcgtcaccg
     3181 taatctgctt gcacgtagat cacataagca ccaagcgcgt tggcctcatg cttgaggaga
     3241 ttgatgagcg cggtggcaat gccctgcctc cggtgctctc cggagactgc gagatcatag
     3301 atatagatct cactacgcgg ctgctcaaac ttgggcagaa cgtaagccgc gagagcgcca
     3361 acaaccgctt cttggtcgaa ggcagcaagc gcgatgaatg tcttactacg gagcaagttc
     3421 ccgaggtaat cggagtccgg ctgatgttgg gagtaggtgg ctacgtctcc gaactcacga
     3481 ccgaaaagat caagagcagc ccgcatggat ttgacttggt cagggccgag cctacatgtg
     3541 cgaatgatgc ccatacttga gccacctaac tttgttttag ggcgactgcc ctgctgcgta
     3601 acatcgttgc tgctgcgtaa catcgttgct gctccataac atcaaacatc gacccacggc
     3661 gtaacgcgct tgctgcttgg atgcccgagg cataggctgt acaaaaaaac agtcataaca
     3721 agccatgaaa accgccactg cgccgttacc accgctgcgt tcggtcaagg ttctggacca
     3781 gttgcgtgag cgcatacgct acttgcatta cagtttacga accgaacagg cttatgtcaa
     3841 gacgtc
//
