LOCUS       40924_6172        5842 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Integrational vector pBELG, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5842)
  AUTHORS   Nikel PI, de Lorenzo V.
  TITLE     Implantation of unmarked regulatory and metabolic modules in 
            Gram-negative bacteria with specialised mini-transposon delivery 
            vectors
  JOURNAL   J. Biotechnol. 163 (2), 143-154 (2013)
  PUBMED    22609234
REFERENCE   2  (bases 1 to 5842)
  AUTHORS   Nikel PI, de Lorenzo V.
  TITLE     Direct Submission
  JOURNAL   Submitted (11-FEB-2012) Systems Biology Program, Centro Nacional de 
            Biotecnologia (CNB-CSIC), C/Darwin 3, Madrid, Madrid 28049, Spain
REFERENCE   3  (bases 1 to 5842)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5842)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. 
            Biotechnol."; date: "2013"; volume: "163"; issue: "2"; pages: 
            "143-154"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (11-FEB-2012) Systems Biology Program, Centro Nacional de 
            Biotecnologia (CNB-CSIC), C/Darwin 3, Madrid, Madrid 28049, Spain"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..19
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase
                     recognition (Reznikoff et al., 2004)"
     promoter        30..107
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             108..1187
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        1422..1451
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    1459..1475
                     /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)."
     misc_feature    1502..1558
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     terminator      1563..1657
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     protein_bind    1668..1715
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(1773..2303)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(2492..2520)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     misc_feature    2521..2568
                     /label=FRT
                     /note="FRT"
     protein_bind    2521..2568
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     misc_feature    complement(2575..2593)
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase
                     recognition (Reznikoff et al., 2004)"
     CDS             2687..4114
                     /codon_start=1
                     /label=Tn5 transposase
                     /note="transposase from the bacterial Tn5 transposon
                     (Reznikoff, 1993)"
                     /translation="MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKS
                     ITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYR
                     HQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESG
                     KWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGL
                     YLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNA
                     VLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAER
                     QRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTP
                     DECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQS
                     KLDGFLAAKDLMAQGIKI"
     promoter        4171..4262
                     /label=AmpR promoter
     CDS             4263..5120
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     terminator      5127..5166
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            5313..5421
                     /label=oriT
                     /note="incP origin of transfer"
     rep_origin      5440..5828
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
ORIGIN
        1 ctgtctctta tacacatctt taattaattg acaccatcga atggtgcaaa acctttcgcg
       61 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
      121 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
      181 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
      241 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
      301 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
      361 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
      421 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
      481 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
      541 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
      601 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
      661 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
      721 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
      781 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
      841 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
      901 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
      961 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     1021 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     1081 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     1141 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     1201 attaatgtga gttagcgcga attgatctgg tttgacagct tatcatcgac tgcacggtgc
     1261 accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa
     1321 atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc
     1381 cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt aatcatccgg
     1441 ctcgtataat gtgtggaatt gtgagcggat aacaatttca caccctaggc cgcggccgcg
     1501 cgaattcgag ctcggtaccc ggggatcctc tagagtcgac ctgcaggcat gcaagcttct
     1561 tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt tgttcagaac
     1621 gctcggttgc cgccgggcgt tttttattgg tgagaatcca gactagtgaa gttcctattc
     1681 cgaagttcct attctctaga aagtatagga acttccaatt taccgaacaa ctccgcggcc
     1741 gggaagccga tctcggcttg aacgaattgt taggtggcgg tacttgggtc gatatcaaag
     1801 tgcatcactt cttcccgtat gcccaacttt gtatagagag ccactgcggg atcgtcaccg
     1861 taatctgctt gcacgtagat cacataagca ccaagcgcgt tggcctcatg cttgaggaga
     1921 ttgatgagcg cggtggcaat gccctgcctc cggtgctctc cggagactgc gagatcatag
     1981 atatagatct cactacgcgg ctgctcaaac ttgggcagaa cgtaagccgc gagagcgcca
     2041 acaaccgctt cttggtcgaa ggcagcaagc gcgatgaatg tcttactacg gagcaagttc
     2101 ccgaggtaat cggagtccgg ctgatgttgg gagtaggtgg ctacgtctcc gaactcacga
     2161 ccgaaaagat caagagcagc ccgcatggat ttgacttggt cagggccgag cctacatgtg
     2221 cgaatgatgc ccatacttga gccacctaac tttgttttag ggcgactgcc ctgctgcgta
     2281 acatcgttgc tgctgcgtaa catcgttgct gctccataac atcaaacatc gacccacggc
     2341 gtaacgcgct tgctgcttgg atgcccgagg cataggctgt acaaaaaaac agtcataaca
     2401 agccatgaaa accgccactg cgccgttacc accgctgcgt tcggtcaagg ttctggacca
     2461 gttgcgtgag cgcatacgct acttgcatta cagtttacga accgaacagg cttatgtcaa
     2521 gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcga cgtcagatgt
     2581 gtataagaga cagctggttt aaactacaca agtagcgtcc tgaacggaac ctttcccgtt
     2641 ttccagaatc tgatgttcca tgtgacctcc taacatggta acgttcatga ttaccagtgc
     2701 actgcatcgt gcggcggatt gggcgaaaag cgtgttttct agtgctgcgc tgggtgatcc
     2761 gcgtcgtacc gcgcgtctgg tgaatgttgc ggcgcaactg gccaaatata gcggcaaaag
     2821 cattaccatt agcagcgaag gcagcaaagc catgcaggaa ggcgcgtatc gttttattcg
     2881 taatccgaac gtgagcgcgg aagcgattcg taaagcgggt gccatgcaga ccgtgaaact
     2941 ggcccaggaa tttccggaac tgctggcaat tgaagatacc acctctctga gctatcgtca
     3001 tcaggtggcg gaagaactgg gcaaactggg tagcattcag gataaaagcc gtggttggtg
     3061 ggtgcatagc gtgctgctgc tggaagcgac cacctttcgt accgtgggcc tgctgcatca
     3121 agaatggtgg atgcgtccgg atgatccggc ggatgcggat gaaaaagaaa gcggcaaatg
     3181 gctggccgct gctgcaactt cgcgtctgag aatgggcagc atgatgagca acgtgattgc
     3241 ggtgtgcgat cgtgaagcgg atattcatgc gtatctgcaa gataaactgg cccataacga
     3301 acgttttgtg gtgcgtagca aacatccgcg taaagatgtg gaaagcggcc tgtatctgta
     3361 tgatcacctg aaaaaccagc cggaactggg cggctatcag attagcattc cgcagaaagg
     3421 cgtggtggat aaacgtggca aacgtaaaaa ccgtccggcg cgtaaagcga gcctgagcct
     3481 gcgtagcggc cgtattaccc tgaaacaggg caacattacc ctgaacgcgg tgctggccga
     3541 agaaattaat ccgccgaaag gcgaaacccc gctgaaatgg ctgctgctga ccagcgagcc
     3601 ggtggaaagt ctggcccaag cgctgcgtgt gattgatatt tatacccatc gttggcgcat
     3661 tgaagaattt cacaaagcgt ggaaaacggg tgcgggtgcg gaacgtcagc gtatggaaga
     3721 accggataac ctggaacgta tggtgagcat tctgagcttt gtggcggtgc gtctgctgca
     3781 actgcgtgaa tcttttactc cgccgcaagc actgcgtgcg cagggcctgc tgaaagaagc
     3841 ggaacacgtt gaaagccaga gcgcggaaac cgtgctgacc ccggatgaat gccaactgct
     3901 gggctatctg gataaaggca aacgcaaacg caaagaaaaa gcgggcagcc tgcaatgggc
     3961 gtatatggcg attgcgcgtc tgggcggctt tatggatagc aaacgtaccg gcattgcgag
     4021 ctggggtgcg ctgtgggaag gttgggaagc gctgcaaagc aaactggatg gctttctggc
     4081 cgcgaaagac ctgatggcgc agggcattaa aatctaatcg atttaaatta gtagcccgcc
     4141 taatgagcgg gctttttttt aattccccta tttgtttatt tttctaaata cattcaaata
     4201 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4261 gtatgagcat tcagcatttt cgtgtggcgc tgattccgtt ttttgcggcg ttttgcctgc
     4321 cggtgtttgc gcatccggaa accctggtga aagtgaaaga tgcggaagat caactgggtg
     4381 cgcgcgtggg ctatattgaa ctggatctga acagcggcaa aattctggaa tcttttcgtc
     4441 cggaagaacg ttttccgatg atgagcacct ttaaagtgct gctgtgcggt gcggttctga
     4501 gccgtgtgga tgcgggccag gaacaactgg gccgtcgtat tcattatagc cagaacgatc
     4561 tggtggaata tagcccggtg accgaaaaac atctgaccga tggcatgacc gtgcgtgaac
     4621 tgtgcagcgc ggcgattacc atgagcgata acaccgcggc gaacctgctg ctgacgacca
     4681 ttggcggtcc gaaagaactg accgcgtttc tgcataacat gggcgatcat gtgacccgtc
     4741 tggatcgttg ggaaccggaa ctgaacgaag cgattccgaa cgatgaacgt gataccacca
     4801 tgccggcagc aatggcgacc accctgcgta aactgctgac gggtgagctg ctgaccctgg
     4861 caagccgcca gcaactgatt gattggatgg aagcggataa agtggcgggt ccgctgctgc
     4921 gtagcgcgct gccggctggc tggtttattg cggataaaag cggtgcgggc gaacgtggca
     4981 gccgtggcat tattgcggcg ctgggcccgg atggtaaacc gagccgtatt gtggtgattt
     5041 ataccaccgg cagccaggcg acgatggatg aacgtaaccg tcagattgcg gaaattggcg
     5101 cgagcctgat taaacattgg taaaccgata caattaaagg ctccttttgg agcctttttt
     5161 tttggacgac ccttgtcctt ttccgctgca taaccctgct tcggggtcat tatagcgatt
     5221 ttttcggtat atccatcctt tttcgcacga tatacaggat tttgccaaag ggttcgtgta
     5281 gactttcctt ggtgtatcca acggcgtcag ccgggcagga taggtgaagt aggcccaccc
     5341 gcgagcgggt gttccttctt cactgtccct tattcgcacc tggcggtgct caacgggaat
     5401 cctgctctgc gaggctggcc gtaggccggc cggcgcgccg atctgaagat cagcagttca
     5461 acctgttgat agtacgtact aagctctcat gtttcacgta ctaagctctc atgtttaacg
     5521 tactaagctc tcatgtttaa cgaactaaac cctcatggct aacgtactaa gctctcatgg
     5581 ctaacgtact aagctctcat gtttcacgta ctaagctctc atgtttgaac aataaaatta
     5641 atataaatca gcaacttaaa tagcctctaa ggttttaagt tttataagaa aaaaaagaat
     5701 atataaggct tttaaagcct ttaaggttta acggttgtgg acaacaagcc agggatgtaa
     5761 cgcactgaga agcccttaga gcctctcaaa gcaattttga gtgacacagg aacacttaac
     5821 ggctgacatg gggcgcgccc ag
//
