LOCUS       Exported                5088 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector DH4-27, complete sequence.
ACCESSION   JX155237
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5088)
  AUTHORS   Huang DC, Holtz WJ, Maharbiz MM.
  TITLE     A genetic bistable switch utilizing nonlinear protein degradation
  JOURNAL   J Biol Eng 6 (1), 9 (2012)
  PUBMED    22776405
REFERENCE   2  (bases 1 to 5088)
  AUTHORS   Huang DC, Holtz WJ, Maharbiz MM.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-JUN-2012) Electrical Engineering and Computer 
            Sciences, University of California, Berkeley, 656 Sutardja Dai Hall,
            Berkeley, CA 94720, USA
REFERENCE   3  (bases 1 to 5088)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: GENtle v. 1.9.4
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5088
                     /organism="Cloning vector DH4-27"
                     /mol_type="other DNA"
                     /db_xref="taxon:1209500"
     regulatory      1..82
                     /regulatory_class="promoter"
                     /note="pRM"
     misc_feature    9..26
                     /label=oR1
                     /note="oR1"
     misc_feature    33..50
                     /label=oR2
                     /note="oR2"
     misc_feature    59..65
                     /label=oR3mut
                     /note="oR3mut"
     5UTR           89..123
     gene            124..921
                     /gene="cI with mf degradation tag"
                     /label=cI with mf degradation tag
     CDS             124..918
                     /codon_start=1
                     /gene="cI with mf degradation tag"
                     /product="CI with mf Lon degradtion tag"
                     /label=cI with mf degradation tag
                     /protein_id="AFN88263.1"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFGAANKNEENTNEVPTFMLNAGQANYAFA"
     CDS             124..834
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=-lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     misc_feature    835..915
                     /gene="cI with mf degradation tag"
                     /label=mf-ssrA tag (LVA)
                     /note="mf-ssrA tag (LVA)"
     5UTR           928..968
     gene            969..1628
                     /gene="TetR"
                     /label=TetR
     CDS             969..1625
                     /codon_start=1
                     /gene="TetR"
                     /product="TetR with LVA degradation tag"
                     /label=TetR
                     /protein_id="AFN88264.1"
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAANDENYALVA"
     CDS             969..1586
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     misc_feature    1590..1622
                     /gene="TetR"
                     /label=LVA tag
                     /note="LVA tag"
     CDS             1590..1622
                     /codon_start=1
                     /product="C-terminal peptide that mediates degradation in 
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
                     /label=ssrA tag (LVA)
                     /note="mutant LVA variant that confers accelerated 
                     degradation under some conditions (Andersen et al., 1998)"
                     /translation="AANDENYALVA"
     regulatory      1653..1781
                     /regulatory_class="terminator"
     terminator      1661..1732
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1748..1775
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      1783..4011
                     /label=pSC101
                     /note="pSC101"
     CDS             complement(2306..3256)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     regulatory      4012..4117
                     /regulatory_class="terminator"
     terminator      4018..4112
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4143..4937)
                     /codon_start=1
                     /product="Neo resistance (Kan)"
                     /label=Neo resistance
                     /protein_id="AFN88265.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"