LOCUS       40924_615        5883 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector DH6-56, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5883)
  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 5883)
  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 5883)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5883)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Eng"; date: "2012"; volume: "6"; issue: "1"; pages: "9"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (10-JUN-2012) Electrical Engineering and Computer Sciences, 
            University of California, Berkeley, 656 Sutardja Dai Hall, Berkeley,
            CA 94720, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: GENtle v. 1.9.4
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(15..36)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(52..1131)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(1132..1209)
                     /label=lacI promoter
     protein_bind    1502..1518
                     /label=lac operator
                     /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)."
     protein_bind    1525..1541
                     /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)."
     5'UTR           1569..1602
     gene            1603..4002
                     /gene="mfLon"
                     /label=mfLon
     CDS             1603..3999
                     /codon_start=1
                     /gene="mfLon"
                     /product="mf-Lon with ssrA degrdation tag LVA"
                     /label=mfLon
                     /protein_id="AFN88256.1"
                     /translation="MSKKIKLPIFQIRGSFIVPGIKENLEVGRKNTLASVNYAIKNSNN
                     QMIAIPQIDASVEKPEFSDLHEFGILIDFEVIKEWKDNSLTISTNPIQRCKVISFFENE
                     DQVPYAEVELIESINDFSDEELKELIEKISDAIKTKASLVTKQIKQLISGESDDLSLAF
                     DSIMFKLAPSKILTNPEYITSPSLKTRWSIIEKIIFAEDGIITRNAESIDAARQKNEIE
                     QELNHKLKEKMDKQQKEYYLREKMRIIKDELEDEDDSDDSSLEKYKERLAKEPFPEEVK
                     RKIMASIKRVEALQSGTPEWNTEKNYIDWMMSIPWWEETEDLTDLKYAKKILDKHHYGM
                     KKVKERIIEYLAVKTKTKSLKAPIITLVGPPGVGKTSLAKSIAEAVGKNFVKVSLGGVK
                     DESEIRGHRKTYVGSMPGRIIQTMKRAKVKNPLFLLDEIDKMASDHRGDPASAMLEVLD
                     PEQNKEFSDHYIEEPYDLSQVMFIATANYPEDIPEALYDRMEIINLSSYTEIEKVKIAQ
                     DYLVPKAIEQHELTSEEISFTEGAINEIIKYYTREAGVRQLERHINSIIRKYIVKNLNG
                     EMDKIVIDEKQVNDLLGKRIFDHTEKQEESQIGVVTGLAYTQFGGDILPIEVSLYPGKG
                     NLILTGKLGEVMKESATIALTYVKSNFEKFGVDKKVFEENDIHVHVPEGAVPKDGPSAG
                     ITITTALISALSDKPVSKEIGMTGEITLRGNVLPIGGLREKSISASRSGLKTIIIPKKN
                     ERDLDEIPDEVKAKLKIIPAEKYEEVFAIVFKTKAANDENYALVA"
     misc_feature    3964..3996
                     /gene="mfLon"
                     /label=degradation tag
                     /note="degradation tag"
     CDS             3964..3996
                     /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"
     terminator      4035..4106
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4122..4149
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(4307..4895)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(4983..5077)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5101..5757)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(5758..5860)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gacgtcggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
       61 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
      121 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
      181 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
      241 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
      301 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
      361 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
      421 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
      481 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
      541 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
      601 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
      661 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
      721 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
      781 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
      841 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
      901 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
      961 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     1021 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     1081 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     1141 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     1201 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     1261 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     1321 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     1381 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     1441 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     1501 attgtgagcg gataacaatt gacattgtga gcggataaca agatactgag cacgaattca
     1561 aaagatctta atatcatcga ccggccggct acgaggataa ggatgagtaa aaaaataaaa
     1621 ctgcctattt tccaaatacg cggttcattt attgttcctg gaattaaaga gaatttagaa
     1681 gttggaagaa aaaacacatt agcgagcgtt aattatgcga ttaaaaattc aaataatcaa
     1741 atgatcgcaa taccgcagat tgatgcatct gttgaaaaac ctgaattttc agatttacat
     1801 gaatttggaa ttttaattga ttttgaagta atcaaagaat ggaaagataa ttcattaact
     1861 attagcacaa accctattca acgatgtaaa gtaattagct tttttgaaaa tgaagatcaa
     1921 gtgccgtatg cggaagttga attaattgaa tcaattaatg attttagtga tgaagagtta
     1981 aaagaattaa ttgaaaaaat ctctgatgct atcaaaacta aagctagttt agtaacaaaa
     2041 caaattaaac agttaatatc aggtgaatct gatgatttga gcttagcttt tgattcaatt
     2101 atgtttaaac tggctccgtc aaaaatattg acaaatccgg aatacataac atctccgtca
     2161 ttaaaaacac gatggtcaat tattgaaaaa ataatttttg ctgaagatgg aatcataaca
     2221 agaaatgcag aatcaattga tgcagcacga caaaagaatg aaattgaaca agaactgaac
     2281 cataaattaa aagaaaaaat ggataaacaa caaaaagaat attatctgcg agaaaaaatg
     2341 cgaattatca aagatgaatt agaagatgaa gatgattctg atgacagttc attagaaaaa
     2401 tataaagaac gtttagctaa agaacctttc cctgaagaag tcaaacgaaa aattatggct
     2461 tctatcaaac gagttgaagc acttcaatca ggaactcctg aatggaatac tgagaaaaat
     2521 tacattgatt ggatgatgag tattccctgg tgggaagaaa ctgaagactt aactgattta
     2581 aaatatgcta aaaaaatttt agacaaacat cattatggta tgaaaaaagt taaagaaaga
     2641 attattgagt acttagctgt taaaactaaa acaaaatcac taaaagcacc aattattaca
     2701 ttagttggtc ccccaggtgt aggtaaaaca agtttagcaa aatcaattgc agaagcagtt
     2761 ggaaaaaact ttgttaaagt aagtttaggt ggagttaaag atgaatcaga aattcgtgga
     2821 catagaaaaa cttatgttgg ttcaatgcca ggaagaatta ttcaaacaat gaaaagagca
     2881 aaagtaaaaa acccattatt cttgcttgat gaaattgata aaatggcatc tgatcacaga
     2941 ggtgatccag caagtgcaat gttagaggtt cttgacccag aacaaaacaa agagttttca
     3001 gatcattata ttgaagaacc atatgatcta agccaagtta tgtttatagc aactgctaac
     3061 tatcctgaag atattcctga agccttatat gaccgtatgg aaatcataaa tttatctagc
     3121 tatactgaaa ttgaaaaagt taagatagca caagattatt tagttccaaa agcaatagaa
     3181 caacatgaat taacttcaga agaaatatca tttactgaag gtgcaattaa tgaaattatt
     3241 aaatattaca ctagagaagc tggtgtgcgt caattagaaa gacatattaa ttctattatt
     3301 agaaagtata ttgttaaaaa tctaaatggt gaaatggata aaattgttat tgatgaaaaa
     3361 caagtaaatg atttattagg taaaagaatt tttgatcaca ctgaaaaaca agaagaatct
     3421 caaattggtg tggttacagg tttagcatat acacaatttg gtggagatat tttaccaatt
     3481 gaagtaagtt tataccctgg taaaggaaat ttaattctta caggtaaact tggtgaagtt
     3541 atgaaagaat ctgcaacaat tgcgttaact tatgttaaat caaactttga aaaatttgga
     3601 gtagataaaa aagtatttga agaaaatgat attcatgttc acgttcctga aggagctgtt
     3661 ccaaaagatg gaccaagtgc tggtatcaca attacaactg ctttaatttc tgcactttca
     3721 gataaaccag tttcaaaaga aattggaatg actggtgaaa tcacactaag aggaaatgta
     3781 ttgccaattg gtggattaag agaaaaatca atttctgctt caagaagtgg attgaaaaca
     3841 atcattattc caaagaaaaa tgaaagagac ttagacgaaa ttccagatga agtaaaagca
     3901 aaactaaaaa ttatacctgc tgaaaaatat gaagaggtat ttgcaatagt ttttaaaaca
     3961 aaagctgcaa acgacgaaaa ctacgcttta gtagcttaat aaggatccaa actcgagtaa
     4021 ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     4081 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc
     4141 ctttctgcgt ttatacctag ggcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4201 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4261 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4321 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4381 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4441 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4501 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4561 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4621 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4681 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4741 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4801 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4861 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4921 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg actagtgctt
     4981 ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg
     5041 agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctcga tatcaaatta
     5101 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg
     5161 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc
     5221 ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac
     5281 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc
     5341 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata
     5401 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc
     5461 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc
     5521 gtctttcatt gccatacgaa attccggatg agcattcatc aggcgggcaa gaatgtgaat
     5581 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc
     5641 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc
     5701 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt
     5761 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat
     5821 ttcattatgg tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccagat
     5881 atc
//
