LOCUS       40924_665        7583 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector DH7-24, complete sequence.
ACCESSION   .
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)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7583)
  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..7583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     5'UTR           complement(2402..2435)
     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    complement(2471..2489)
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      2496..2577
                     /label=pRM
                     /note="pRM"
                     /regulatory_class="promoter"
     5'UTR           2584..2618
     CDS             2619..3329
                     /label=lambda repressor
                     /note="phage lambda repressor"
     misc_feature    3330..3410
                     /gene="cI-mf-ssra tag"
                     /label=mf-Lon ssrA degradation tag
                     /note="mf-Lon ssrA degradation tag"
     5'UTR           3423..3463
     CDS             3464..4084
                     /label=TetR
                     /note="tetracycline repressor TetR"
     CDS             4085..4117
                     /label=ssrA tag (LVA)
                     /note="C-terminal peptide that mediates degradation in
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
     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"
     CDS             complement(4804..5751)
                     /label=Rep101
                     /note="RepA protein needed for replication with the pSC101 
                     origin"
     rep_origin      complement(5799..6021)
                     /direction=LEFT
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(6513..6607)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6641..7432)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
ORIGIN
        1 ttattaagct actaaagcgt agttttcgtc gtttgcagct tttgttttaa aaactattgc
       61 aaatacctct tcatattttt cagcaggtat aatttttagt tttgctttta cttcatctgg
      121 aatttcgtct aagtctcttt catttttctt tggaataatg attgttttca atccacttct
      181 tgaagcagaa attgattttt ctcttaatcc accaattggc aatacatttc ctcttagtgt
      241 gatttcacca gtcattccaa tttcttttga aactggttta tctgaaagtg cagaaattaa
      301 agcagttgta attgtgatac cagcacttgg tccatctttt ggaacagctc cttcaggaac
      361 gtgaacatga atatcatttt cttcaaatac ttttttatct actccaaatt tttcaaagtt
      421 tgatttaaca taagttaacg caattgttgc agattctttc ataacttcac caagtttacc
      481 tgtaagaatt aaatttcctt taccagggta taaacttact tcaattggta aaatatctcc
      541 accaaattgt gtatatgcta aacctgtaac cacaccaatt tgagattctt cttgtttttc
      601 agtgtgatca aaaattcttt tacctaataa atcatttact tgtttttcat caataacaat
      661 tttatccatt tcaccattta gatttttaac aatatacttt ctaataatag aattaatatg
      721 tctttctaat tgacgcacac cagcttctct agtgtaatat ttaataattt cattaattgc
      781 accttcagta aatgatattt cttctgaagt taattcatgt tgttctattg cttttggaac
      841 taaataatct tgtgctatct taactttttc aatttcagta tagctagata aatttatgat
      901 ttccatacgg tcatataagg cttcaggaat atcttcagga tagttagcag ttgctataaa
      961 cataacttgg cttagatcat atggttcttc aatataatga tctgaaaact ctttgttttg
     1021 ttctgggtca agaacctcta acattgcact tgctggatca cctctgtgat cagatgccat
     1081 tttatcaatt tcatcaagca agaataatgg gttttttact tttgctcttt tcattgtttg
     1141 aataattctt cctggcattg aaccaacata agtttttcta tgtccacgaa tttctgattc
     1201 atctttaact ccacctaaac ttactttaac aaagtttttt ccaactgctt ctgcaattga
     1261 ttttgctaaa cttgttttac ctacacctgg gggaccaact aatgtaataa ttggtgcttt
     1321 tagtgatttt gttttagttt taacagctaa gtactcaata attctttctt taactttttt
     1381 cataccataa tgatgtttgt ctaaaatttt tttagcatat tttaaatcag ttaagtcttc
     1441 agtttcttcc caccagggaa tactcatcat ccaatcaatg taatttttct cagtattcca
     1501 ttcaggagtt cctgattgaa gtgcttcaac tcgtttgata gaagccataa tttttcgttt
     1561 gacttcttca gggaaaggtt ctttagctaa acgttcttta tatttttcta atgaactgtc
     1621 atcagaatca tcttcatctt ctaattcatc tttgataatt cgcatttttt ctcgcagata
     1681 atattctttt tgttgtttat ccattttttc ttttaattta tggttcagtt cttgttcaat
     1741 ttcattcttt tgtcgtgctg catcaattga ttctgcattt cttgttatga ttccatcttc
     1801 agcaaaaatt attttttcaa taattgacca tcgtgttttt aatgacggag atgttatgta
     1861 ttccggattt gtcaatattt ttgacggagc cagtttaaac ataattgaat caaaagctaa
     1921 gctcaaatca tcagattcac ctgatattaa ctgtttaatt tgttttgtta ctaaactagc
     1981 tttagttttg atagcatcag agattttttc aattaattct tttaactctt catcactaaa
     2041 atcattaatt gattcaatta attcaacttc cgcatacggc acttgatctt cattttcaaa
     2101 aaagctaatt actttacatc gttgaatagg gtttgtgcta atagttaatg aattatcttt
     2161 ccattctttg attacttcaa aatcaattaa aattccaaat tcatgtaaat ctgaaaattc
     2221 aggtttttca acagatgcat caatctgcgg tattgcgatc atttgattat ttgaattttt
     2281 aatcgcataa ttaacgctcg ctaatgtgtt ttttcttcca acttctaaat tctctttaat
     2341 tccaggaaca ataaatgaac cgcgtatttg gaaaataggc agttttattt ttttactcat
     2401 accagcacct ccttcttctc cggtcgatga tattagtgct cagtatctct atcactgata
     2461 gggatgtcaa tctctatcac tgatagggag gatctgcaac cattatcacc gccagaggta
     2521 aaatagtcaa cacgcacggt gttagatatt tataaatagt ggtgatagat ttaacgtgga
     2581 tctaatccaa caatatagca tactctcgcg acggcctcat gagcacaaaa aagaaaccat
     2641 taacacaaga gcagcttgag gacgcacgtc gccttaaagc aatttatgaa aaaaagaaaa
     2701 atgaacttgg cttatcccag gaatctgtcg cagacaagat ggggatgggg cagtcaggcg
     2761 ttggtgcttt atttaatggc atcaatgcat taaatgctta taacgccgca ttgcttgcaa
     2821 aaattctcaa agttagcgtt gaagaattta gcccttcaat cgccagagaa atctacgaga
     2881 tgtatgaagc ggttagtatg cagccgtcac ttagaagtga gtatgagtac cctgtttttt
     2941 ctcatgttca ggcagggatg ttctcacctg agcttagaac ctttaccaaa ggtgatgcgg
     3001 agagatgggt aagcacaacc aaaaaagcca gtgattctgc attctggctt gaggttgaag
     3061 gtaattccat gaccgcacca acaggctcca agccaagctt tcctgacgga atgttaattc
     3121 tcgttgaccc tgagcaggct gttgagccag gtgatttctg catagccaga cttgggggtg
     3181 atgagtttac cttcaagaaa ctgatcaggg atagcggtca ggtgttttta caaccactaa
     3241 acccacagta cccaatgatc ccatgcaatg agagttgttc cgttgtgggg aaagttatcg
     3301 ctagtcagtg gcctgaagag acgtttggcg cggcgaataa aaatgaagaa aataccaatg
     3361 aagtgccgac ctttatgctg aatgcgggcc aggcgaatta tgcgtttgcg taataaggat
     3421 ctcaagccgc agctaaaagg taggatatac aagaccagga tctatgtcca gattagataa
     3481 aagtaaagtg attaacagcg cattagagct gcttaatgag gtcggaatcg aaggtttaac
     3541 aacccgtaaa ctcgcccaga agctaggtgt agagcagcct acattgtatt ggcatgtaaa
     3601 aaataagcgg gctttgctcg acgccttagc cattgagatg ttagataggc accatactca
     3661 cttttgccct ttagaagggg aaagctggca agatttttta cgtaataacg ctaaaagttt
     3721 tagatgtgct ttactaagtc atcgcgatgg agcaaaagta catttaggta cacggcctac
     3781 agaaaaacag tatgaaactc tcgaaaatca attagccttt ttatgccaac aaggtttttc
     3841 actagagaat gcattatatg cactcagcgc tgtggggcat tttactttag gttgcgtatt
     3901 ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa acacctacta ctgatagtat
     3961 gccgccatta ttacgacaag ctatcgaatt atttgatcac caaggtgcag agccagcctt
     4021 cttattcggc cttgaattga tcatatgcgg attagaaaaa caacttaaat gtgaaagtgg
     4081 gtccgctgca aacgacgaaa actacgcttt agtagcttaa taaggatcct aactcgagta
     4141 aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     4201 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     4261 cctttctgcg tttataccta gggtacgggt tttgctgccc gcaaacgggc tgttctggtg
     4321 ttgctagttt gttatcagaa tcgcagatcc ggcttcagcc ggtttgccgg ctgaaagcgc
     4381 tatttcttcc agaattgcca tgattttttc cccacgggag gcgtcactgg ctcccgtgtt
     4441 gtcggcagct ttgattcgat aagcagcatc gcctgtttca ggctgtctat gtgtgactgt
     4501 tgagctgtaa caagttgtct caggtgttca atttcatgtt ctagttgctt tgttttactg
     4561 gtttcacctg ttctattagg tgttacatgc tgttcatctg ttacattgtc gatctgttca
     4621 tggtgaacag ctttgaatgc accaaaaact cgtaaaagct ctgatgtatc tatctttttt
     4681 acaccgtttt catctgtgca tatggacagt tttccctttg atatgtaacg gtgaacagtt
     4741 gttctacttt tgtttgttag tcttgatgct tcactgatag atacaagagc cataagaacc
     4801 tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt tttgcgtgag
     4861 ccatgagaac gaaccattga gatcatactt actttgcatg tcactcaaaa attttgcctc
     4921 aaaactggtg agctgaattt ttgcagttaa agcatcgtgt agtgtttttc ttagtccgtt
     4981 atgtaggtag gaatctgatg taatggttgt tggtattttg tcaccattca tttttatctg
     5041 gttgttctca agttcggtta cgagatccat ttgtctatct agttcaactt ggaaaatcaa
     5101 cgtatcagtc gggcggcctc gcttatcaac caccaatttc atattgctgt aagtgtttaa
     5161 atctttactt attggtttca aaacccattg gttaagcctt ttaaactcat ggtagttatt
     5221 ttcaagcatt aacatgaact taaattcatc aaggctaatc tctatatttg ccttgtgagt
     5281 tttcttttgt gttagttctt ttaataacca ctcataaatc ctcatagagt atttgttttc
     5341 aaaagactta acatgttcca gattatattt tatgaatttt tttaactgga aaagataagg
     5401 caatatctct tcactaaaaa ctaattctaa tttttcgctt gagaacttgg catagtttgt
     5461 ccactggaaa atctcaaagc ctttaaccaa aggattcctg atttccacag ttctcgtcat
     5521 cagctctctg gttgctttag ctaatacacc ataagcattt tccctactga tgttcatcat
     5581 ctgagcgtat tggttataag tgaacgatac cgtccgttct ttccttgtag ggttttcaat
     5641 cgtggggttg agtagtgcca cacagcataa aattagcttg gtttcatgct ccgttaagtc
     5701 atagcgacta atcgctagtt catttgcttt gaaaacaact aattcagaca tacatctcaa
     5761 ttggtctagg tgattttaat cactatacca attgagatgg gctagtcaat gataattact
     5821 agtccttttc ccgggtgatc tgggtatctg taaattctgc tagacctttg ctggaaaact
     5881 tgtaaattct gctagaccct ctgtaaattc cgctagacct ttgtgtgttt tttttgttta
     5941 tattcaagtg gttataattt atagaataaa gaaagaataa aaaaagataa aaagaataga
     6001 tcccagccct gtgtataact cactacttta gtcagttccg cagtattaca aaaggatgtc
     6061 gcaaacgctg tttgctcctc tacaaaacag accttaaaac cctaaaggct taagtagcac
     6121 cctcgcaagc tcgggcaaat cgctgaatat tccttttgtc tccgaccatc aggcacctga
     6181 gtcgctgtct ttttcgtgac attcagttcg ctgcgctcac ggctctggca gtgaatgggg
     6241 gtaaatggca ctacaggcgc cttttatgga ttcatgcaag gaaactaccc ataatacaag
     6301 aaaagcccgt cacgggcttc tcagggcgtt ttatggcggg tctgctatgt ggtgctatct
     6361 gactttttgc tgttcagcag ttcctgccct ctgattttcc agtctgacca cttcggatta
     6421 tcccgtgaca ggtcattcag actggctaat gcacccagta aggcagcggt atcatcaaca
     6481 ggcttacccg tcttactgtc cctagtgctt ggattctcac caataaaaaa cgcccggcgg
     6541 caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac
     6601 aggagtccaa gcgagctctc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc
     6661 gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt
     6721 cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat
     6781 agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca
     6841 ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca
     6901 tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca
     6961 gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt
     7021 tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat
     7081 cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg
     7141 gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg
     7201 cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat
     7261 tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc
     7321 ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc
     7381 tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg
     7441 atcctcatcc tgtctcttga tcagatcatg atcccctgcg ccatcagatc cttggcggca
     7501 agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg
     7561 gcaattccga attcatgaga tct
//
