LOCUS       Exported                8684 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pET15b-vioB, complete sequence.
ACCESSION   KX461960
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8684)
  AUTHORS   Pardee K, Slomovic S, Nguyen PQ, Lee JW, Donghia N, Burrill D, 
            Ferrante T, McSorley F, Furuta Y, Lewandowski M, Boddy CN, Joshi NS,
            Collins JJ.
  TITLE     Portable, On-demand Biomolecular Manufacturing
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8684)
  AUTHORS   Pardee K, Slomovic S, Nguyen PQ, Lee JW, Donghia N, Burrill D, 
            Ferrante T, McSorley F, Furuta Y, Lewandowski M, Boddy CN, Joshi NS,
            Collins JJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (29-JUN-2016) Wyss Institute, Harvard University, 3 
            Blackfan Circle, Wyss Institute Rm517-1D, Boston, MA 02115, USA
REFERENCE   3  (bases 1 to 8684)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8684
                     /organism="cloning vector pET15b-vioB"
                     /mol_type="other DNA"
                     /db_xref="taxon:1898573"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             209..1069
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     CDS             407..1069
                     /codon_start=1
                     /product="AmpR"
                     /label=AmpR
                     /protein_id="AON96392.1"
                     /translation="MSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHL
                     TDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEA
                     IPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFI
                     ADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     rep_origin      1164..1846
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2014..2156
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2258..2449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3761..4843)
                     /codon_start=1
                     /product="LacIq"
                     /label=LacIq
                     /protein_id="AON96393.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(4844..4921)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        5230..5248
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5230..5245
                     /regulatory_class="promoter"
                     /note="T7"
     protein_bind    5249..5273
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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    5249..5271
                     /label=lacO operator site
                     /note="lacO operator site"
     misc_feature    5257
                     /label=transcription start
                     /note="transcription start"
     RBS             5288..5310
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      5305..5310
                     /regulatory_class="ribosome_binding_site"
                     /note="RBS"
     CDS             5317..8346
                     /codon_start=1
                     /product="VioB"
                     /label=VioB
                     /note="derived from Chromobacterium violaceum"
                     /protein_id="AON96394.1"
                     /translation="MSILDFPRIHFRGWARVNAPTANRDPHGHIDMASNTVAMAGEPFD
                     LARHPTEFHRHLRSLGPRFGLDGRADPEGPFSLAEGYNAAGNNHFSWESATVSHVQWDG
                     GEADRGDGLVGARLALWGHYNDYLRTTFNRARWVDSDPTRRDAAQIYAGQFTISPAGAG
                     PGTPWLFTADIDDSHGARWTRGGHIAERGGHFLDEEFGLARLFQFSVPKDHPHFLFHPG
                     PFDSEAWRRLQLALEDDDVLGLTVQYALFNMSTPPQPNSPVFHDMVGVVGLWRRGELAS
                     YPAGRLLRPRQPGLGDLTLRVNGGRVALNLACAIPFSTRAAQPSAPDRLTPDLGAKLPL
                     GDLLLRDEDGALLARVPQALYQDYWTNHGIVDLPLLREPRGSLTLSSELAEWREQDWVT
                     QSDASNLYLEAPDRRHGRFFPESIALRSYFRGEARARPDIPHRIEGMGLVGVESRQDGD
                     AAEWRLTGLRPGPARIVLDDGAEAIPLRVLPDDWALDDATVEEVDYAFLYRHVMAYYEL
                     VYPFMSDKVFSLADRCKCETYARLMWQMCDPQNRNKSYYMPSTRELSAPKARLFLKYLA
                     HVEGQARLQAPPPAGPARIESKAQLAAELRKAVDLELSVMLQYLYAAYSIPNYAQGQQR
                     VRDGAWTAEQLQLACGSGDRRRDGGIRAALLEIAHEEMIHYLVVNNLLMALGEPFYAGV
                     PLMGEAARQAFGLDTEFALEPFSESTLARFVRLEWPHFIPAPGKSIADCYAAIRQAFLD
                     LPDLFGGEAGKRGGEHHLFLNELTNRAHPGYQLEVFDRDSALFGIAFVTDQGEGGALDS
                     PHYEHSHFQRLREMSARIMAQSAPFEPALPALRNPVLDESPGCQRVADGRARALMALYQ
                     GVYELMFAMMAQHFAVKPLGSLRRSRLMNAAIDLMTGLLRPLSCALMNLPSGIAGRTAG
                     PPLPGPVDTRSYDDYALGCRMLARRCERLLEQASMLEPGWLPDAQMELLDFYRRQMLDL
                     ACGKLSREATSGDYKDDDDK"
     misc_feature    8320..8343
                     /label=FLAG tag
                     /note="FLAG tag"
     CDS             8320..8343
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      8425..8472
                     /regulatory_class="terminator"
                     /note="T7 terminator"
     terminator      8425..8472
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(8647..8675)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"