LOCUS       40924_6177        5964 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Integrational vector pBELK, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5964)
  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 5964)
  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 5964)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5964)
  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..5964
                     /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(1729..2541)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     misc_feature    2643..2690
                     /label=FRT
                     /note="FRT"
     protein_bind    2643..2690
                     /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(2697..2715)
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase
                     recognition (Reznikoff et al., 2004)"
     CDS             2809..4236
                     /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        4293..4384
                     /label=AmpR promoter
     CDS             4385..5242
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     terminator      5249..5288
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            5435..5543
                     /label=oriT
                     /note="incP origin of transfer"
     rep_origin      5562..5950
                     /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 aattattaga aaaattcatc
     1741 cagcatcaga tgaaattgca gtttgttcat atccggatta tcaatgccat atttctgaaa
     1801 cagacgtttt tgcaggctcg ggctaaattc gcccaggcag ttccacagaa tggccagatc
     1861 ctgataacga tccgcaatgc ccacacggcc cacatcaatg cagccaatca gtttgccttc
     1921 atcgaaaatc aggttatcca ggctaaaatc gccgtgggtc accacgctat ccgggctaaa
     1981 cggcagcagt ttatgcattt ctttccacac ctgttccacc ggccagccgt tacgttcatc
     2041 atcaaaatcg ctcgcatcca ccaggccgtt gttcatacgg ctctgcgcct gggccagacg
     2101 aaacacacga tcgctgttaa acgggcagtt gcacaccgga atgctatgca gacgacgcag
     2161 aaacacggcc agcgcatcca caatgttttc gccgctatcc ggatattctt ccagcacctg
     2221 aaacgcggtt ttgcccggaa tcgcggtggt cagcagccac gcatcatccg gggtgcgaat
     2281 aaaatgttta atggtcggca gcggcataaa ttcggtcagc cagttcagac gcaccatttc
     2341 atcggtcaca tcgttcgcca cgctgccttt gccatgtttc agaaacagtt ccggcgcatc
     2401 cggtttgcca tacagacgat aaatggtcgc gccgctctga cccacgttat cacgcgccca
     2461 tttatagcca tacagatccg catccatgtt gctgttcaga cgcggacggc tacagctcgt
     2521 ttcacgctga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     2581 tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac
     2641 aagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc gacgtcagat
     2701 gtgtataaga gacagctggt ttaaactaca caagtagcgt cctgaacgga acctttcccg
     2761 ttttccagaa tctgatgttc catgtgacct cctaacatgg taacgttcat gattaccagt
     2821 gcactgcatc gtgcggcgga ttgggcgaaa agcgtgtttt ctagtgctgc gctgggtgat
     2881 ccgcgtcgta ccgcgcgtct ggtgaatgtt gcggcgcaac tggccaaata tagcggcaaa
     2941 agcattacca ttagcagcga aggcagcaaa gccatgcagg aaggcgcgta tcgttttatt
     3001 cgtaatccga acgtgagcgc ggaagcgatt cgtaaagcgg gtgccatgca gaccgtgaaa
     3061 ctggcccagg aatttccgga actgctggca attgaagata ccacctctct gagctatcgt
     3121 catcaggtgg cggaagaact gggcaaactg ggtagcattc aggataaaag ccgtggttgg
     3181 tgggtgcata gcgtgctgct gctggaagcg accacctttc gtaccgtggg cctgctgcat
     3241 caagaatggt ggatgcgtcc ggatgatccg gcggatgcgg atgaaaaaga aagcggcaaa
     3301 tggctggccg ctgctgcaac ttcgcgtctg agaatgggca gcatgatgag caacgtgatt
     3361 gcggtgtgcg atcgtgaagc ggatattcat gcgtatctgc aagataaact ggcccataac
     3421 gaacgttttg tggtgcgtag caaacatccg cgtaaagatg tggaaagcgg cctgtatctg
     3481 tatgatcacc tgaaaaacca gccggaactg ggcggctatc agattagcat tccgcagaaa
     3541 ggcgtggtgg ataaacgtgg caaacgtaaa aaccgtccgg cgcgtaaagc gagcctgagc
     3601 ctgcgtagcg gccgtattac cctgaaacag ggcaacatta ccctgaacgc ggtgctggcc
     3661 gaagaaatta atccgccgaa aggcgaaacc ccgctgaaat ggctgctgct gaccagcgag
     3721 ccggtggaaa gtctggccca agcgctgcgt gtgattgata tttataccca tcgttggcgc
     3781 attgaagaat ttcacaaagc gtggaaaacg ggtgcgggtg cggaacgtca gcgtatggaa
     3841 gaaccggata acctggaacg tatggtgagc attctgagct ttgtggcggt gcgtctgctg
     3901 caactgcgtg aatcttttac tccgccgcaa gcactgcgtg cgcagggcct gctgaaagaa
     3961 gcggaacacg ttgaaagcca gagcgcggaa accgtgctga ccccggatga atgccaactg
     4021 ctgggctatc tggataaagg caaacgcaaa cgcaaagaaa aagcgggcag cctgcaatgg
     4081 gcgtatatgg cgattgcgcg tctgggcggc tttatggata gcaaacgtac cggcattgcg
     4141 agctggggtg cgctgtggga aggttgggaa gcgctgcaaa gcaaactgga tggctttctg
     4201 gccgcgaaag acctgatggc gcagggcatt aaaatctaat cgatttaaat tagtagcccg
     4261 cctaatgagc gggctttttt ttaattcccc tatttgttta tttttctaaa tacattcaaa
     4321 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4381 gagtatgagc attcagcatt ttcgtgtggc gctgattccg ttttttgcgg cgttttgcct
     4441 gccggtgttt gcgcatccgg aaaccctggt gaaagtgaaa gatgcggaag atcaactggg
     4501 tgcgcgcgtg ggctatattg aactggatct gaacagcggc aaaattctgg aatcttttcg
     4561 tccggaagaa cgttttccga tgatgagcac ctttaaagtg ctgctgtgcg gtgcggttct
     4621 gagccgtgtg gatgcgggcc aggaacaact gggccgtcgt attcattata gccagaacga
     4681 tctggtggaa tatagcccgg tgaccgaaaa acatctgacc gatggcatga ccgtgcgtga
     4741 actgtgcagc gcggcgatta ccatgagcga taacaccgcg gcgaacctgc tgctgacgac
     4801 cattggcggt ccgaaagaac tgaccgcgtt tctgcataac atgggcgatc atgtgacccg
     4861 tctggatcgt tgggaaccgg aactgaacga agcgattccg aacgatgaac gtgataccac
     4921 catgccggca gcaatggcga ccaccctgcg taaactgctg acgggtgagc tgctgaccct
     4981 ggcaagccgc cagcaactga ttgattggat ggaagcggat aaagtggcgg gtccgctgct
     5041 gcgtagcgcg ctgccggctg gctggtttat tgcggataaa agcggtgcgg gcgaacgtgg
     5101 cagccgtggc attattgcgg cgctgggccc ggatggtaaa ccgagccgta ttgtggtgat
     5161 ttataccacc ggcagccagg cgacgatgga tgaacgtaac cgtcagattg cggaaattgg
     5221 cgcgagcctg attaaacatt ggtaaaccga tacaattaaa ggctcctttt ggagcctttt
     5281 tttttggacg acccttgtcc ttttccgctg cataaccctg cttcggggtc attatagcga
     5341 ttttttcggt atatccatcc tttttcgcac gatatacagg attttgccaa agggttcgtg
     5401 tagactttcc ttggtgtatc caacggcgtc agccgggcag gataggtgaa gtaggcccac
     5461 ccgcgagcgg gtgttccttc ttcactgtcc cttattcgca cctggcggtg ctcaacggga
     5521 atcctgctct gcgaggctgg ccgtaggccg gccggcgcgc cgatctgaag atcagcagtt
     5581 caacctgttg atagtacgta ctaagctctc atgtttcacg tactaagctc tcatgtttaa
     5641 cgtactaagc tctcatgttt aacgaactaa accctcatgg ctaacgtact aagctctcat
     5701 ggctaacgta ctaagctctc atgtttcacg tactaagctc tcatgtttga acaataaaat
     5761 taatataaat cagcaactta aatagcctct aaggttttaa gttttataag aaaaaaaaga
     5821 atatataagg cttttaaagc ctttaaggtt taacggttgt ggacaacaag ccagggatgt
     5881 aacgcactga gaagccctta gagcctctca aagcaatttt gagtgacaca ggaacactta
     5941 acggctgaca tggggcgcgc ccag
//
