LOCUS       40924_645        7579 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector DH7-20, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7579)
  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 7579)
  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 7579)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7579)
  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..7579
                     /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="AFN88293.1"
                     /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..2431)
     protein_bind    2442..2460
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    complement(2467..2485)
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      2492..2573
                     /label=pRM
                     /note="pRM"
                     /regulatory_class="promoter"
     5'UTR           2580..2614
     CDS             2615..3325
                     /label=lambda repressor
                     /note="phage lambda repressor"
     misc_feature    3326..3406
                     /gene="cI-mf-ssra tag"
                     /label=mf-Lon ssrA degradation tag
                     /note="mf-Lon ssrA degradation tag"
     5'UTR           3419..3459
     CDS             3460..4080
                     /label=TetR
                     /note="tetracycline repressor TetR"
     CDS             4081..4113
                     /label=ssrA tag (LVA)
                     /note="C-terminal peptide that mediates degradation in
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
     terminator      4152..4223
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4239..4266
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4800..5747)
                     /label=Rep101
                     /note="RepA protein needed for replication with the pSC101 
                     origin"
     rep_origin      complement(5795..6017)
                     /direction=LEFT
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(6509..6603)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6637..7428)
                     /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 ccttatctta gccggccggt cgatgatatt agtgctcagt atctctatca ctgataggga
     2461 tgtcaatctc tatcactgat agggaggatc tgcaaccatt atcaccgcca gaggtaaaat
     2521 agtcaacacg cacggtgtta gatatttata aatagtggtg atagatttaa cgtggatcta
     2581 atccaacaat atagcatact ctcgcgacgg cctcatgagc acaaaaaaga aaccattaac
     2641 acaagagcag cttgaggacg cacgtcgcct taaagcaatt tatgaaaaaa agaaaaatga
     2701 acttggctta tcccaggaat ctgtcgcaga caagatgggg atggggcagt caggcgttgg
     2761 tgctttattt aatggcatca atgcattaaa tgcttataac gccgcattgc ttgcaaaaat
     2821 tctcaaagtt agcgttgaag aatttagccc ttcaatcgcc agagaaatct acgagatgta
     2881 tgaagcggtt agtatgcagc cgtcacttag aagtgagtat gagtaccctg ttttttctca
     2941 tgttcaggca gggatgttct cacctgagct tagaaccttt accaaaggtg atgcggagag
     3001 atgggtaagc acaaccaaaa aagccagtga ttctgcattc tggcttgagg ttgaaggtaa
     3061 ttccatgacc gcaccaacag gctccaagcc aagctttcct gacggaatgt taattctcgt
     3121 tgaccctgag caggctgttg agccaggtga tttctgcata gccagacttg ggggtgatga
     3181 gtttaccttc aagaaactga tcagggatag cggtcaggtg tttttacaac cactaaaccc
     3241 acagtaccca atgatcccat gcaatgagag ttgttccgtt gtggggaaag ttatcgctag
     3301 tcagtggcct gaagagacgt ttggcgcggc gaataaaaat gaagaaaata ccaatgaagt
     3361 gccgaccttt atgctgaatg cgggccaggc gaattatgcg tttgcgtaat aaggatctca
     3421 agccgcagct aaaaggtagg atatacaaga ccaggatcta tgtccagatt agataaaagt
     3481 aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg tttaacaacc
     3541 cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca tgtaaaaaat
     3601 aagcgggctt tgctcgacgc cttagccatt gagatgttag ataggcacca tactcacttt
     3661 tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa aagttttaga
     3721 tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg gcctacagaa
     3781 aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg tttttcacta
     3841 gagaatgcat tatatgcact cagcgctgtg gggcatttta ctttaggttg cgtattggaa
     3901 gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga tagtatgccg
     3961 ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc agccttctta
     4021 ttcggccttg aattgatcat atgcggatta gaaaaacaac ttaaatgtga aagtgggtcc
     4081 gctgcaaacg acgaaaacta cgctttagta gcttaataag gatcctaact cgagtaagga
     4141 tctccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     4201 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
     4261 tctgcgttta tacctagggt acgggttttg ctgcccgcaa acgggctgtt ctggtgttgc
     4321 tagtttgtta tcagaatcgc agatccggct tcagccggtt tgccggctga aagcgctatt
     4381 tcttccagaa ttgccatgat tttttcccca cgggaggcgt cactggctcc cgtgttgtcg
     4441 gcagctttga ttcgataagc agcatcgcct gtttcaggct gtctatgtgt gactgttgag
     4501 ctgtaacaag ttgtctcagg tgttcaattt catgttctag ttgctttgtt ttactggttt
     4561 cacctgttct attaggtgtt acatgctgtt catctgttac attgtcgatc tgttcatggt
     4621 gaacagcttt gaatgcacca aaaactcgta aaagctctga tgtatctatc ttttttacac
     4681 cgttttcatc tgtgcatatg gacagttttc cctttgatat gtaacggtga acagttgttc
     4741 tacttttgtt tgttagtctt gatgcttcac tgatagatac aagagccata agaacctcag
     4801 atccttccgt atttagccag tatgttctct agtgtggttc gttgtttttg cgtgagccat
     4861 gagaacgaac cattgagatc atacttactt tgcatgtcac tcaaaaattt tgcctcaaaa
     4921 ctggtgagct gaatttttgc agttaaagca tcgtgtagtg tttttcttag tccgttatgt
     4981 aggtaggaat ctgatgtaat ggttgttggt attttgtcac cattcatttt tatctggttg
     5041 ttctcaagtt cggttacgag atccatttgt ctatctagtt caacttggaa aatcaacgta
     5101 tcagtcgggc ggcctcgctt atcaaccacc aatttcatat tgctgtaagt gtttaaatct
     5161 ttacttattg gtttcaaaac ccattggtta agccttttaa actcatggta gttattttca
     5221 agcattaaca tgaacttaaa ttcatcaagg ctaatctcta tatttgcctt gtgagttttc
     5281 ttttgtgtta gttcttttaa taaccactca taaatcctca tagagtattt gttttcaaaa
     5341 gacttaacat gttccagatt atattttatg aattttttta actggaaaag ataaggcaat
     5401 atctcttcac taaaaactaa ttctaatttt tcgcttgaga acttggcata gtttgtccac
     5461 tggaaaatct caaagccttt aaccaaagga ttcctgattt ccacagttct cgtcatcagc
     5521 tctctggttg ctttagctaa tacaccataa gcattttccc tactgatgtt catcatctga
     5581 gcgtattggt tataagtgaa cgataccgtc cgttctttcc ttgtagggtt ttcaatcgtg
     5641 gggttgagta gtgccacaca gcataaaatt agcttggttt catgctccgt taagtcatag
     5701 cgactaatcg ctagttcatt tgctttgaaa acaactaatt cagacataca tctcaattgg
     5761 tctaggtgat tttaatcact ataccaattg agatgggcta gtcaatgata attactagtc
     5821 cttttcccgg gtgatctggg tatctgtaaa ttctgctaga cctttgctgg aaaacttgta
     5881 aattctgcta gaccctctgt aaattccgct agacctttgt gtgttttttt tgtttatatt
     5941 caagtggtta taatttatag aataaagaaa gaataaaaaa agataaaaag aatagatccc
     6001 agccctgtgt ataactcact actttagtca gttccgcagt attacaaaag gatgtcgcaa
     6061 acgctgtttg ctcctctaca aaacagacct taaaacccta aaggcttaag tagcaccctc
     6121 gcaagctcgg gcaaatcgct gaatattcct tttgtctccg accatcaggc acctgagtcg
     6181 ctgtcttttt cgtgacattc agttcgctgc gctcacggct ctggcagtga atgggggtaa
     6241 atggcactac aggcgccttt tatggattca tgcaaggaaa ctacccataa tacaagaaaa
     6301 gcccgtcacg ggcttctcag ggcgttttat ggcgggtctg ctatgtggtg ctatctgact
     6361 ttttgctgtt cagcagttcc tgccctctga ttttccagtc tgaccacttc ggattatccc
     6421 gtgacaggtc attcagactg gctaatgcac ccagtaaggc agcggtatca tcaacaggct
     6481 tacccgtctt actgtcccta gtgcttggat tctcaccaat aaaaaacgcc cggcggcaac
     6541 cgagcgttct gaacaaatcc agatggagtt ctgaggtcat tactggatct atcaacagga
     6601 gtccaagcga gctctcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata
     6661 gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc
     6721 ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg
     6781 gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat
     6841 gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg
     6901 cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc
     6961 atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc
     7021 ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc
     7081 catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac
     7141 ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca
     7201 aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag
     7261 ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa
     7321 cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc
     7381 cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc
     7441 tcatcctgtc tcttgatcag atcatgatcc cctgcgccat cagatccttg gcggcaagaa
     7501 agccatccag tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa
     7561 ttccgaattc atgagatct
//
