LOCUS       40924_655        7582 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector DH7-22, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7582)
  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 7582)
  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 7582)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7582)
  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..7582
                     /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="AFN88301.1"
                     /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(2401..2434)
     protein_bind    2445..2463
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    complement(2470..2488)
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      2495..2576
                     /label=pRM
                     /note="pRM"
                     /regulatory_class="promoter"
     5'UTR           2583..2617
     CDS             2618..3328
                     /label=lambda repressor
                     /note="phage lambda repressor"
     misc_feature    3329..3409
                     /gene="cI-mf-ssra tag"
                     /label=mf-Lon ssrA degradation tag
                     /note="mf-Lon ssrA degradation tag"
     5'UTR           3422..3462
     CDS             3463..4083
                     /label=TetR
                     /note="tetracycline repressor TetR"
     CDS             4084..4116
                     /label=ssrA tag (LVA)
                     /note="C-terminal peptide that mediates degradation in
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
     terminator      4155..4226
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4242..4269
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4803..5750)
                     /label=Rep101
                     /note="RepA protein needed for replication with the pSC101 
                     origin"
     rep_origin      complement(5798..6020)
                     /direction=LEFT
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(6512..6606)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6640..7431)
                     /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 ccttatcctc gtagccggcc ggtcgatgat attagtgctc agtatctcta tcactgatag
     2461 ggatgtcaat ctctatcact gatagggagg atctgcaacc attatcaccg ccagaggtaa
     2521 aatagtcaac acgcacggtg ttagatattt ataaatagtg gtgatagatt taacgtggat
     2581 ctaatccaac aatatagcat actctcgcga cggcctcatg agcacaaaaa agaaaccatt
     2641 aacacaagag cagcttgagg acgcacgtcg ccttaaagca atttatgaaa aaaagaaaaa
     2701 tgaacttggc ttatcccagg aatctgtcgc agacaagatg gggatggggc agtcaggcgt
     2761 tggtgcttta tttaatggca tcaatgcatt aaatgcttat aacgccgcat tgcttgcaaa
     2821 aattctcaaa gttagcgttg aagaatttag cccttcaatc gccagagaaa tctacgagat
     2881 gtatgaagcg gttagtatgc agccgtcact tagaagtgag tatgagtacc ctgttttttc
     2941 tcatgttcag gcagggatgt tctcacctga gcttagaacc tttaccaaag gtgatgcgga
     3001 gagatgggta agcacaacca aaaaagccag tgattctgca ttctggcttg aggttgaagg
     3061 taattccatg accgcaccaa caggctccaa gccaagcttt cctgacggaa tgttaattct
     3121 cgttgaccct gagcaggctg ttgagccagg tgatttctgc atagccagac ttgggggtga
     3181 tgagtttacc ttcaagaaac tgatcaggga tagcggtcag gtgtttttac aaccactaaa
     3241 cccacagtac ccaatgatcc catgcaatga gagttgttcc gttgtgggga aagttatcgc
     3301 tagtcagtgg cctgaagaga cgtttggcgc ggcgaataaa aatgaagaaa ataccaatga
     3361 agtgccgacc tttatgctga atgcgggcca ggcgaattat gcgtttgcgt aataaggatc
     3421 tcaagccgca gctaaaaggt aggatataca agaccaggat ctatgtccag attagataaa
     3481 agtaaagtga ttaacagcgc attagagctg cttaatgagg tcggaatcga aggtttaaca
     3541 acccgtaaac tcgcccagaa gctaggtgta gagcagccta cattgtattg gcatgtaaaa
     3601 aataagcggg ctttgctcga cgccttagcc attgagatgt tagataggca ccatactcac
     3661 ttttgccctt tagaagggga aagctggcaa gattttttac gtaataacgc taaaagtttt
     3721 agatgtgctt tactaagtca tcgcgatgga gcaaaagtac atttaggtac acggcctaca
     3781 gaaaaacagt atgaaactct cgaaaatcaa ttagcctttt tatgccaaca aggtttttca
     3841 ctagagaatg cattatatgc actcagcgct gtggggcatt ttactttagg ttgcgtattg
     3901 gaagatcaag agcatcaagt cgctaaagaa gaaagggaaa cacctactac tgatagtatg
     3961 ccgccattat tacgacaagc tatcgaatta tttgatcacc aaggtgcaga gccagccttc
     4021 ttattcggcc ttgaattgat catatgcgga ttagaaaaac aacttaaatg tgaaagtggg
     4081 tccgctgcaa acgacgaaaa ctacgcttta gtagcttaat aaggatccta actcgagtaa
     4141 ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     4201 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc
     4261 ctttctgcgt ttatacctag ggtacgggtt ttgctgcccg caaacgggct gttctggtgt
     4321 tgctagtttg ttatcagaat cgcagatccg gcttcagccg gtttgccggc tgaaagcgct
     4381 atttcttcca gaattgccat gattttttcc ccacgggagg cgtcactggc tcccgtgttg
     4441 tcggcagctt tgattcgata agcagcatcg cctgtttcag gctgtctatg tgtgactgtt
     4501 gagctgtaac aagttgtctc aggtgttcaa tttcatgttc tagttgcttt gttttactgg
     4561 tttcacctgt tctattaggt gttacatgct gttcatctgt tacattgtcg atctgttcat
     4621 ggtgaacagc tttgaatgca ccaaaaactc gtaaaagctc tgatgtatct atctttttta
     4681 caccgttttc atctgtgcat atggacagtt ttccctttga tatgtaacgg tgaacagttg
     4741 ttctactttt gtttgttagt cttgatgctt cactgataga tacaagagcc ataagaacct
     4801 cagatccttc cgtatttagc cagtatgttc tctagtgtgg ttcgttgttt ttgcgtgagc
     4861 catgagaacg aaccattgag atcatactta ctttgcatgt cactcaaaaa ttttgcctca
     4921 aaactggtga gctgaatttt tgcagttaaa gcatcgtgta gtgtttttct tagtccgtta
     4981 tgtaggtagg aatctgatgt aatggttgtt ggtattttgt caccattcat ttttatctgg
     5041 ttgttctcaa gttcggttac gagatccatt tgtctatcta gttcaacttg gaaaatcaac
     5101 gtatcagtcg ggcggcctcg cttatcaacc accaatttca tattgctgta agtgtttaaa
     5161 tctttactta ttggtttcaa aacccattgg ttaagccttt taaactcatg gtagttattt
     5221 tcaagcatta acatgaactt aaattcatca aggctaatct ctatatttgc cttgtgagtt
     5281 ttcttttgtg ttagttcttt taataaccac tcataaatcc tcatagagta tttgttttca
     5341 aaagacttaa catgttccag attatatttt atgaattttt ttaactggaa aagataaggc
     5401 aatatctctt cactaaaaac taattctaat ttttcgcttg agaacttggc atagtttgtc
     5461 cactggaaaa tctcaaagcc tttaaccaaa ggattcctga tttccacagt tctcgtcatc
     5521 agctctctgg ttgctttagc taatacacca taagcatttt ccctactgat gttcatcatc
     5581 tgagcgtatt ggttataagt gaacgatacc gtccgttctt tccttgtagg gttttcaatc
     5641 gtggggttga gtagtgccac acagcataaa attagcttgg tttcatgctc cgttaagtca
     5701 tagcgactaa tcgctagttc atttgctttg aaaacaacta attcagacat acatctcaat
     5761 tggtctaggt gattttaatc actataccaa ttgagatggg ctagtcaatg ataattacta
     5821 gtccttttcc cgggtgatct gggtatctgt aaattctgct agacctttgc tggaaaactt
     5881 gtaaattctg ctagaccctc tgtaaattcc gctagacctt tgtgtgtttt ttttgtttat
     5941 attcaagtgg ttataattta tagaataaag aaagaataaa aaaagataaa aagaatagat
     6001 cccagccctg tgtataactc actactttag tcagttccgc agtattacaa aaggatgtcg
     6061 caaacgctgt ttgctcctct acaaaacaga ccttaaaacc ctaaaggctt aagtagcacc
     6121 ctcgcaagct cgggcaaatc gctgaatatt ccttttgtct ccgaccatca ggcacctgag
     6181 tcgctgtctt tttcgtgaca ttcagttcgc tgcgctcacg gctctggcag tgaatggggg
     6241 taaatggcac tacaggcgcc ttttatggat tcatgcaagg aaactaccca taatacaaga
     6301 aaagcccgtc acgggcttct cagggcgttt tatggcgggt ctgctatgtg gtgctatctg
     6361 actttttgct gttcagcagt tcctgccctc tgattttcca gtctgaccac ttcggattat
     6421 cccgtgacag gtcattcaga ctggctaatg cacccagtaa ggcagcggta tcatcaacag
     6481 gcttacccgt cttactgtcc ctagtgcttg gattctcacc aataaaaaac gcccggcggc
     6541 aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca
     6601 ggagtccaag cgagctctcg aaccccagag tcccgctcag aagaactcgt caagaaggcg
     6661 atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc
     6721 agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata
     6781 gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac
     6841 catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat
     6901 gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag
     6961 atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt
     7021 cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc
     7081 agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg
     7141 cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc
     7201 gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt
     7261 cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg
     7321 gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct
     7381 ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga
     7441 tcctcatcct gtctcttgat cagatcatga tcccctgcgc catcagatcc ttggcggcaa
     7501 gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg
     7561 caattccgaa ttcatgagat ct
//
