LOCUS       40924_43813        4692 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pToxin-mfLON, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4692)
  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 4692)
  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 4692)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4692)
  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..4692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      7..80
                     /label=pLscrO-2
                     /note="pLscrO-2"
                     /regulatory_class="promoter"
     regulatory      81..102
                     /label=RBS
                     /note="RBS"
                     /regulatory_class="ribosome_binding_site"
     CDS             103..2547
                     /codon_start=1
                     /product="mfLON protease"
                     /label=mfLON protease
                     /protein_id="ALP32109.1"
                     /translation="MSKKIKLPIFQIRGSFIVPGIKENLEVGRKNTLASVNYAIKNSNN
                     QMIAIPQIDASVEKPEFSDLHEFGILIDFEVIKEWKDNSLTISTNPIQRCKVISFFENE
                     DQVPYAEVELIESINDFSDEELKELIEKISDAIKTKASLVTKQIKQLISGESDDLSLAF
                     DSIMFKLAPSKILTNPEYITSPSLKTRWSIIEKIIFAEDGIITRNAESIDAARQKNEIE
                     QELNHKLKEKMDKQQKEYYLREKMRIIKDELEDEDDSDDSSLEKYKERLAKEPFPEEVK
                     RKIMASIKRVEALQSGTPEWNTEKNYIDWMMSIPWWEETEDLTDLKYAKKILDKHHYGM
                     KKVKERIIEYLAVKTKTKSLKAPIITLVGPPGVGKTSLAKSIAEAVGKNFVKVSLGGVK
                     DESEIRGHRKTYVGSMPGRIIQTMKRAKVKNPLFLLDEIDKMASDHRGDPASAMLEVLD
                     PEQNKEFSDHYIEEPYDLSQVMFIATANYPEDIPEALYDRMEIINLSSYTEIEKVKIAQ
                     DYLVPKAIEQHELTSEEISFTEGAINEIIKYYTREAGVRQLERHINSIIRKYIVKNLNG
                     EMDKIVIDEKQVNDLLGKRIFDHTEKQEESQIGVVTGLAYTQFGGDILPIEVSLYPGKG
                     NLILTGKLGEVMKESATIALTYVKSNFEKFGVDKKVFEENDIHVHVPEGAVPKDGPSAG
                     ITITTALISALSDKPVSKEIGMTGEITLRGNVLPIGGLREKSISASRSGLKTIIIPKKN
                     ERDLDEIPDEVKAKLKIIPAEKYEEVFAIVFKTKAANKNEENTNEVPTFMLNAGQANRR
                     RV"
     terminator      2589..2675
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     rep_origin      2796..3341
                     /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(3503..3597)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3630..4487)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4488..4592)
                     /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 cttgatgggg gatcccatgg tacgcgtgct
     2581 agaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     2641 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gccctagacc tagtaacaac
     2701 ttatatcgta tggggctgac ttcaggtgct acatttgaag agataaattg cactgaaatc
     2761 tagaaatatt ttatctgatt aataagatga tcttcttgag atcgttttgg tctgcgcgta
     2821 atctcttgct ctgaaaacga aaaaaccgcc ttgcagggcg gtttttcgaa ggttctctga
     2881 gctaccaact ctttgaaccg aggtaactgg cttggaggag cgcagtcacc aaaacttgtc
     2941 ctttcagttt agccttaacc ggcgcatgac ttcaagacta actcctctaa atcaattacc
     3001 agtggctgct gccagtggtg cttttgcatg tctttccggg ttggactcaa gacgatagtt
     3061 accggataag gcgcagcggt cggactgaac ggggggttcg tgcatacagt ccagcttgga
     3121 gcgaactgcc tacccggaac tgagtgtcag gcgtggaatg agacaaacgc ggccataaca
     3181 gcggaatgac accggtaaac cgaaaggcag gaacaggaga gcgcacgagg gagccgccag
     3241 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccaccactga tttgagcgtc
     3301 agatttcgtg atgcttgtca ngggggcgga gcctatggaa aaacggcttt gccgcggccc
     3361 tctcacttcc ctgttaagta tcttcctggc atcttccagg aaatctccgc cccgttcgta
     3421 agccatttcc gctcgccgca gtcgaacgac cgagcgtagc gagtcagtga gcgaggaagc
     3481 ggaatatatc cctagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg
     3541 ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa
     3601 gcgagctcgt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3661 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3721 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3781 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3841 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3901 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3961 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4021 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4081 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4141 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4201 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4261 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4321 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4381 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4441 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4501 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4561 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     4621 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     4681 tttcgtcttc ac
//
