LOCUS       Exported                5729 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pToxin-mfLON-ecoRI, complete sequence.
ACCESSION   KT895277
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)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5729
                     /organism="Cloning vector pToxin-mfLON-ecoRI"
                     /mol_type="other DNA"
                     /db_xref="taxon:1758747"
     regulatory      7..54
                     /regulatory_class="promoter"
                     /note="pLscrO-2"
     regulatory      81..101
                     /regulatory_class="ribosome_binding_site"
                     /note="RBS"
     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
                     /regulatory_class="terminator"
                     /note="T0"
     terminator      2561..2655
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     regulatory      2662..2709
                     /regulatory_class="promoter"
                     /note="pLscrO-2"
     regulatory      2742..2761
                     /regulatory_class="ribosome_binding_site"
                     /note="RBS"
     CDS             2768..3601
                     /codon_start=1
                     /product="type-2 restriction enzyme EcoRI"
                     /label=type-2 restriction enzyme EcoRI
                     /protein_id="ALP32114.1"
                     /translation="MSNKKQSNRLTEQHKLSQGVIGIFGDYAKAHDLAVGEVSKLVKKA
                     LSNEYPQLSFRYRDSIKKTEINEALKKIDPDLGGTLFVSNSSIKPDGGIVEVKDDYGEW
                     RVVLVAEAKHQGKDIINIRNGLLVGKRGDQDLMAAGNAIERSHKNISEIANFMLSESHF
                     PYVLFLEGSNFLTENISITRPDGRVVNLEYNSGILNRLDRLTAANYGMPINSNLCINKF
                     VNHKDKSIMLQAASIYTQGDGREWDSKIMFEIMFDISTTSLRVLGRDLFEQLTSK"
     regulatory      3621..3680
                     /regulatory_class="terminator"
                     /note="T1"
     terminator      3626..3712
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      3732..4528
                     /label=p15A
                     /note="p15A"
     rep_origin      3833..4378
                     /direction=RIGHT
                     /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      4540..4634
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4664..5524)
                     /codon_start=1
                     /product="beta-lactmase"
                     /label=beta-lactmase
                     /protein_id="ALP32115.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5525..5629)
                     /gene="bla"
                     /label=AmpR promoter