LOCUS       40924_43808        5729 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pToxin-mfLON-ecoRI, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5729)
  AUTHORS   Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ.
  TITLE     'Deadman' and 'Passcode' microbial kill switches for bacterial 
            containment
  JOURNAL   Nat. Chem. Biol. 12 (2), 82-86 (2016)
  PUBMED    26641934
REFERENCE   2  (bases 1 to 5729)
  AUTHORS   Chan CTY., Lee JW, Cameron DE, Bashor CJ, Collins JJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (12-OCT-2015) Department of Biological Engineering, 
            Massachusetts Institute of Technology, 77 Massachusetts Ave., 
            E25-302b, Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 5729)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5729)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. Chem. 
            Biol."; date: "2016"; volume: "12"; issue: "2"; pages: "82-86"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (12-OCT-2015) Department of Biological Engineering, Massachusetts 
            Institute of Technology, 77 Massachusetts Ave., E25-302b, Cambridge,
            MA 02139, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      7..54
                     /label=pLscrO-2
                     /note="pLscrO-2"
                     /regulatory_class="promoter"
     regulatory      81..101
                     /label=RBS
                     /note="RBS"
                     /regulatory_class="ribosome_binding_site"
     CDS             103..2547
                     /codon_start=1
                     /product="mfLON protease"
                     /label=mfLON protease
                     /protein_id="ALP32113.1"
                     /translation="MSKKIKLPIFQIRGSFIVPGIKENLEVGRKNTLASVNYAIKNSNN
                     QMIAIPQIDASVEKPEFSDLHEFGILIDFEVIKEWKDNSLTISTNPIQRCKVISFFENE
                     DQVPYAEVELIESINDFSDEELKELIEKISDAIKTKASLVTKQIKQLISGESDDLSLAF
                     DSIMFKLAPSKILTNPEYITSPSLKTRWSIIEKIIFAEDGIITRNAESIDAARQKNEIE
                     QELNHKLKEKMDKQQKEYYLREKMRIIKDELEDEDDSDDSSLEKYKERLAKEPFPEEVK
                     RKIMASIKRVEALQSGTPEWNTEKNYIDWMMSIPWWEETEDLTDLKYAKKILDKHHYGM
                     KKVKERIIEYLAVKTKTKSLKAPIITLVGPPGVGKTSLAKSIAEAVGKNFVKVSLGGVK
                     DESEIRGHRKTYVGSMPGRIIQTMKRAKVKNPLFLLDEIDKMASDHRGDPASAMLEVLD
                     PEQNKEFSDHYIEEPYDLSQVMFIATANYPEDIPEALYDRMEIINLSSYTEIEKVKIAQ
                     DYLVPKAIEQHELTSEEISFTEGAINEIIKYYTREAGVRQLERHINSIIRKYIVKNLNG
                     EMDKIVIDEKQVNDLLGKRIFDHTEKQEESQIGVVTGLAYTQFGGDILPIEVSLYPGKG
                     NLILTGKLGEVMKESATIALTYVKSNFEKFGVDKKVFEENDIHVHVPEGAVPKDGPSAG
                     ITITTALISALSDKPVSKEIGMTGEITLRGNVLPIGGLREKSISASRSGLKTIIIPKKN
                     ERDLDEIPDEVKAKLKIIPAEKYEEVFAIVFKTKAANKNEENTNEVPTFMLNAGQANRR
                     RV"
     regulatory      2561..2655
                     /label=T0
                     /note="T0"
                     /regulatory_class="terminator"
     terminator      2561..2655
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     regulatory      2662..2709
                     /label=pLscrO-2
                     /note="pLscrO-2"
                     /regulatory_class="promoter"
     regulatory      2742..2761
                     /label=RBS
                     /note="RBS"
                     /regulatory_class="ribosome_binding_site"
     CDS             2768..3598
                     /gene="ecoRIR"
                     /label=ecoRIR
                     /note="Type II restriction enzyme EcoRI from Escherichia
                     coli. Accession#: P00642"
     terminator      3626..3712
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     rep_origin      3833..4378
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     terminator      complement(4540..4634)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4667..5524)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5525..5629)
                     /label=AmpR promoter
ORIGIN
        1 ctcgagcatt tattaaaccg gtttattgac ataaaccggt ttagcataga tactgagcac
       61 atcagcagga cgcactgacc gaattcgtat ctaggaggta ccatgagtaa aaaaatcaaa
      121 ctacctattt tccaaatccg tggttctttc atcgttccgg gtatcaaaga aaacctggaa
      181 gttggtcgta aaaacaccct ggcttctgtt aactacgcta tcaaaaactc taacaaccag
      241 atgatcgcta tcccgcagat cgacgcttct gttgaaaaac cggagttctc tgacctgcac
      301 gagttcggta tcctgatcga cttcgaagtt atcaaagaat ggaaagacaa ctctctgacc
      361 atctctacca acccgatcca gcgttgcaaa gttatctctt tcttcgaaaa cgaagaccag
      421 gttccgtacg ctgaagttga actgatcgaa tctatcaacg acttctctga cgaagaactg
      481 aaagaactga tcgaaaaaat ctctgacgct atcaaaacca aggcttctct ggttaccaaa
      541 cagatcaaac aactgatctc tggtgaatct gacgacctgt ctctggcttt cgactctatc
      601 atgttcaaac tggctccgtc taaaatcctg accaacccgg aatacatcac ctctccgtct
      661 ctgaaaaccc gttggtctat catcgaaaaa atcatcttcg ctgaagacgg tatcatcacc
      721 cgtaacgctg aatctatcga cgctgctcgt cagaaaaacg aaatcgaaca ggaactgaac
      781 cacaaactga aagaaaaaat ggacaaacag cagaaagaat actacctgcg tgaaaaaatg
      841 cgtatcatca aagacgaact ggaagacgaa gacgactctg acgactcttc tctggaaaaa
      901 tacaaagaac gtctggctaa agaaccgttc ccggaagaag ttaaacgtaa aatcatggct
      961 tctatcaaac gtgttgaagc tctgcaatct ggaaccccgg aatggaacac cgaaaaaaac
     1021 tacatcgact ggatgatgtc tatcccgtgg tgggaagaaa ccgaagacct gaccgacctg
     1081 aaatacgcta aaaaaatcct ggacaaacac cactacggta tgaaaaaagt taaagaacgt
     1141 atcatcgaat acctggctgt taaaaccaaa accaaatctc tgaaagctcc gatcatcacc
     1201 ctggttggtc cgccgggtgt tggtaaaacc tctctggcta aatctatcgc tgaagctgtt
     1261 ggtaaaaact tcgttaaagt ttctctgggt ggtgttaaag acgaatctga aatccgtggt
     1321 caccgtaaaa cctacgttgg ttctatgccg ggtcgtatca tccagaccat gaaacgtgct
     1381 aaagttaaaa acccgctgtt cctgctggac gaaatcgaca aaatggcttc tgaccaccgt
     1441 ggtgacccgg cttctgctat gctggaagtt ctggacccgg aacagaacaa agagttctct
     1501 gaccactaca tcgaagaacc gtacgacctg tctcaggtta tgttcatcgc taccgctaac
     1561 tacccggaag acatcccgga agctctgtac gaccgtatgg aaatcatcaa cctgtcttct
     1621 tacaccgaaa tcgaaaaagt taaaatcgct caggactacc tggttccgaa agctatcgaa
     1681 cagcacgaac tgacctctga agaaatctct ttcaccgaag gtgctatcaa cgaaatcatc
     1741 aaatactaca cccgtgaagc tggtgttcgt caactggaac gtcacatcaa ctctatcatc
     1801 cgtaaataca tcgttaaaaa cctgaacggt gaaatggaca aaatcgttat cgacgaaaaa
     1861 caggttaacg acctgctggg taaacgtatc ttcgaccaca ccgaaaaaca ggaagaatct
     1921 cagatcggtg ttgttaccgg tctggcttac acccagttcg gtggtgacat cctgccgatt
     1981 gaagtttctc tgtatccggg taaaggtaac ctgatcctga ccggtaaact gggtgaagtt
     2041 atgaaagaat ctgctaccat cgctctgacc tacgttaaat ctaacttcga aaaattcggt
     2101 gttgacaaaa aagttttcga agaaaacgac atccacgttc acgttccgga aggtgctgtt
     2161 ccgaaagacg gtccgtctgc tggtatcacc atcaccaccg ctctgatctc tgctctgtct
     2221 gacaaaccgg tttctaaaga aatcggtatg accggtgaaa tcaccctgcg tggtaacgtt
     2281 ctgccgattg gtggtctgcg tgaaaaatct atctctgctt ctcgttctgg tctgaaaacc
     2341 atcatcatcc cgaaaaaaaa cgaacgtgac ctggacgaaa tcccggacga agttaaagct
     2401 aaactgaaaa tcatcccggc tgaaaaatac gaagaggtat ttgcaatagt ttttaaaaca
     2461 aaagcggcga acaaaaacga agaaaacacc aacgaagtgc cgacctttat gctgaacgcg
     2521 ggccaggcga acagaagacg agtttaaaag ctttgcttgg attctcacca ataaaaaacg
     2581 cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat
     2641 ctatcaacag gagtccggcc gcatttatta aaccggttta ttgacataaa ccggtttagc
     2701 atagatactg agcacatcag caggacgcac tgacccaatt gtcaggaata ggcccgtcgc
     2761 cggatccatg tctaataaaa aacagtcaaa taggctaact gaacaacata agttatctca
     2821 aggtgtaatt gggatttttg gggattatgc aaaagctcat gatctcgctg ttggtgaggt
     2881 ttcaaaatta gtaaagaaag ctcttagcaa cgaataccct caattatcat ttcgatatag
     2941 agatagtata aagaaaacag aaataaatga ggctttaaaa aaaattgacc ctgatcttgg
     3001 cggtacttta tttgtttcaa attccagcat caaacctgat ggtggaattg tagaggtcaa
     3061 agatgattat ggtgaatgga gagttgtact tgttgctgaa gccaaacacc aaggtaaaga
     3121 tattataaat ataaggaatg gtttgttagt tgggaaaaga ggagatcaag atttaatggc
     3181 tgctggtaat gctatcgaaa gatctcataa gaatatatca gagatagcga attttatgct
     3241 ctctgagagc cactttcctt acgtcctttt cttagagggg tctaactttt taacagaaaa
     3301 tatctcaata acaagaccag atggaagggt tgttaatctt gagtataatt ctggtatatt
     3361 aaataggtta gatcgactaa cagcggctaa ttatggaatg cctataaata gtaatctatg
     3421 tattaacaaa tttgtaaatc ataaagacaa aagcattatg ctacaagcag catctatata
     3481 tactcaagga gatgggaggg agtgggattc gaaaatcatg tttgaaataa tgtttgatat
     3541 atcaacgact tcgctcagag tgttggggcg tgacttgttt gaacagctta catctaagtg
     3601 accatggtac gcgtgctaga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     3661 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccgcc
     3721 ctagacctag taacaactta tatcgtatgg ggctgacttc aggtgctaca tttgaagaga
     3781 taaattgcac tgaaatctag aaatatttta tctgattaat aagatgatct tcttgagatc
     3841 gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg cagggcggtt
     3901 tttcgaaggt tctctgagct accaactctt tgaaccgagg taactggctt ggaggagcgc
     3961 agtcaccaaa acttgtcctt tcagtttagc cttaaccggc gcatgacttc aagactaact
     4021 cctctaaatc aattaccagt ggctgctgcc agtggtgctt ttgcatgtct ttccgggttg
     4081 gactcaagac gatagttacc ggataaggcg cagcggtcgg actgaacggg gggttcgtgc
     4141 atacagtcca gcttggagcg aactgcctac ccggaactga gtgtcaggcg tggaatgaga
     4201 caaacgcggc cataacagcg gaatgacacc ggtaaaccga aaggcaggaa caggagagcg
     4261 cacgagggag ccgccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4321 ccactgattt gagcgtcaga tttcgtgatg cttgtcangg gggcggagcc tatggaaaaa
     4381 cggctttgcc gcggccctct cacttccctg ttaagtatct tcctggcatc ttccaggaaa
     4441 tctccgcccc gttcgtaagc catttccgct cgccgcagtc gaacgaccga gcgtagcgag
     4501 tcagtgagcg aggaagcgga atatatccct agtgcttgga ttctcaccaa taaaaaacgc
     4561 ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc
     4621 tatcaacagg agtccaagcg agctcgtaaa cttggtctga cagttaccaa tgcttaatca
     4681 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4741 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     4801 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4861 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4921 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4981 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5041 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5101 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5161 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5221 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5281 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5341 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5401 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5461 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5521 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     5581 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     5641 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     5701 ggcgtatcac gaggcccttt cgtcttcac
//
