LOCUS       Exported                7583 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector DH7-24, complete sequence.
ACCESSION   JX155253
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7583)
  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 7583)
  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 7583)
  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..7583
                     /organism="Cloning vector DH7-24"
                     /mol_type="other DNA"
                     /db_xref="taxon:1209516"
     gene            complement(1..2400)
                     /gene="mf-lon with LVA"
                     /label=mf-lon with LVA
     CDS             complement(4..2400)
                     /codon_start=1
                     /gene="mf-lon with LVA"
                     /product="mf Lon with LVA degradation tag"
                     /label=mf-lon with LVA
                     /protein_id="AFN88309.1"
                     /translation="MSKKIKLPIFQIRGSFIVPGIKENLEVGRKNTLASVNYAIKNSNN
                     QMIAIPQIDASVEKPEFSDLHEFGILIDFEVIKEWKDNSLTISTNPIQRCKVISFFENE
                     DQVPYAEVELIESINDFSDEELKELIEKISDAIKTKASLVTKQIKQLISGESDDLSLAF
                     DSIMFKLAPSKILTNPEYITSPSLKTRWSIIEKIIFAEDGIITRNAESIDAARQKNEIE
                     QELNHKLKEKMDKQQKEYYLREKMRIIKDELEDEDDSDDSSLEKYKERLAKEPFPEEVK
                     RKIMASIKRVEALQSGTPEWNTEKNYIDWMMSIPWWEETEDLTDLKYAKKILDKHHYGM
                     KKVKERIIEYLAVKTKTKSLKAPIITLVGPPGVGKTSLAKSIAEAVGKNFVKVSLGGVK
                     DESEIRGHRKTYVGSMPGRIIQTMKRAKVKNPLFLLDEIDKMASDHRGDPASAMLEVLD
                     PEQNKEFSDHYIEEPYDLSQVMFIATANYPEDIPEALYDRMEIINLSSYTEIEKVKIAQ
                     DYLVPKAIEQHELTSEEISFTEGAINEIIKYYTREAGVRQLERHINSIIRKYIVKNLNG
                     EMDKIVIDEKQVNDLLGKRIFDHTEKQEESQIGVVTGLAYTQFGGDILPIEVSLYPGKG
                     NLILTGKLGEVMKESATIALTYVKSNFEKFGVDKKVFEENDIHVHVPEGAVPKDGPSAG
                     ITITTALISALSDKPVSKEIGMTGEITLRGNVLPIGGLREKSISASRSGLKTIIIPKKN
                     ERDLDEIPDEVKAKLKIIPAEKYEEVFAIVFKTKAANDENYALVA"
     misc_feature    complement(7..39)
                     /gene="mf-lon with LVA"
                     /label=degradation tag (LVA)
                     /note="degradation tag (LVA)"
     CDS             complement(7..39)
                     /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"
     5UTR           complement(2402..2435)
     regulatory      complement(2436..2489)
                     /regulatory_class="promoter"
                     /note="pLtetO-1"
     protein_bind    2446..2464
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2471..2489
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      2496..2577
                     /regulatory_class="promoter"
                     /note="pRM"
     5UTR           2584..2618
     gene            2619..3416
                     /gene="cI-mf-ssra tag"
                     /label=cI-mf-ssra tag
     CDS             2619..3413
                     /codon_start=1
                     /gene="cI-mf-ssra tag"
                     /product="CI with mf Lon degradation tag"
                     /label=cI-mf-ssra tag
                     /protein_id="AFN88310.1"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFGAANKNEENTNEVPTFMLNAGQANYAFA"
     CDS             2619..3329
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=-lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     misc_feature    3330..3410
                     /gene="cI-mf-ssra tag"
                     /label=mf-Lon ssrA degradation tag
                     /note="mf-Lon ssrA degradation tag"
     5UTR           3423..3463
     gene            3464..4123
                     /gene="TetR ssra tag"
                     /label=TetR ssra tag
     CDS             3464..4120
                     /codon_start=1
                     /gene="TetR ssra tag"
                     /product="TetR with LVA degradation tag"
                     /label=TetR ssra tag
                     /protein_id="AFN88311.1"
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAANDENYALVA"
     CDS             3464..4081
                     /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    4085..4117
                     /gene="TetR ssra tag"
                     /label=degradation tag (LVA)
                     /note="degradation tag (LVA)"
     CDS             4085..4117
                     /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      4148..4276
                     /regulatory_class="terminator"
     terminator      4156..4227
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4243..4270
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      4278..6506
                     /label=pSC101
                     /note="pSC101"
     CDS             complement(4801..5751)
                     /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      6507..6612
                     /regulatory_class="terminator"
     terminator      6513..6607
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6638..7432)
                     /codon_start=1
                     /product="Neo resistance (Kan)"
                     /label=Neo resistance
                     /protein_id="AFN88312.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"