LOCUS       sequence-172537-        4153 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the cDHFR-link2-His6-MBP- NleG6-3 EHEC in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4153)
  AUTHORS   Levin-Kravets O, Tanner N, Shohat N, Attali I, Keren-Kaplan T, 
            Shusterman A, Artzi S, Varvak A, Reshef Y, Shi X, Zucker O, Baram T,
            Katina C, Pilzer I, Ben-Aroya S, Prag G
  TITLE     A bacterial genetic selection system for ubiquitylation cascade 
            discovery.
  JOURNAL   Nat Methods. 2016 Nov;13(11):945-952. doi: 10.1038/nmeth.4003. Epub 
            2016 Oct 3.
  PUBMED    27694912
REFERENCE   2  (bases 1 to 4153)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nmeth.4003"; journalName: "Nat Methods"; date: "2016-11"; 
            volume: "13"; issue: "11"; pages: "945-952"
COMMENT     Sequence Label: pCD-Sub69-NleG6-3 EHEC
FEATURES             Location/Qualifiers
     source          1..4153
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     114..132
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(153..171,172..177,178..194,195..200,201..226)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    153..171
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    178..196
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             236..247
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             620..637
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             656..1753
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1727..1750
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1814..1837
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(2485..3276)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(3277..3368)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(3416..4153,1))
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
ORIGIN
        1 cttttctact gaaccgctct agatttcagt gcaatttatc tcttcaaatg tagcaccgtc
       61 gacgcaattc cgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
      121 gtatcacgag gccctttcgt cttcacctcg agtccctatc agtgatagag attgacatcc
      181 ctatcagtga tagagatact gagcacatca gcaggacgca ctgaccgaat tcattaaaga
      241 ggagaaaacg cgtatgagta aagtagacat ggtttggata gtcggaggca gttctgttta
      301 ccaggaagcc atgaatcaac caggccacct cagactcttt gtgacaagga tcatgcagga
      361 atttgaaagt gacacgtttt tcccagaaat tgatttgggg aaatataaac ttctcccaga
      421 atacccaggc gtcctctctg aggtccagga ggaaaaaggc atcaagtata agtttgaagt
      481 ctacgagaag aaagacaggc gcgccggcgg ctcagcgggc tcgggaagcg gagcgggatc
      541 tggcagcggc ggtagcgcag gcagcagcgg gtcaagcggt gcctcgagcg gcaggcgcgc
      601 cctgcagatg ggcagcagcc atcaccatca tcaccacagc caggatccga attcgaaaat
      661 cgaagaaggt aaactggtaa tctggattaa cggcgataaa ggctataacg gtctcgctga
      721 agtcggtaag aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa
      781 actggaagag aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg
      841 ggcacacgac cgctttggtg gctacgctca atctggcctg ttggctgaaa tcaccccgga
      901 caaagcgttc caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa
      961 gctgattgct tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct
     1021 gccgaacccg ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa
     1081 aggtaagagc gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc
     1141 tgctgacggg ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg
     1201 cgtggataac gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa
     1261 acacatgaat gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac
     1321 agcgatgacc atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta
     1381 tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct
     1441 gagcgcaggt attaacgccg ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa
     1501 ctatctgctg actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt
     1561 agcgctgaag tcttacgagg aagagttggc gaaagatcca cgtattgccg ccacaatgga
     1621 aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc
     1681 cgtgcgtact gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa
     1741 agacgcgcag actaattcga gctcgaacaa caacaacaat aacaataaca acaacctcgg
     1801 gatcgatacg accctggaag ttctgttcca ggggcccccg cggatggaga gaagggctgt
     1861 ggctctggaa aggcaattaa atggaggtgt cgatttttta aggagtgtta ataactattt
     1921 tcagagtgtc atggcagaac acagagaaaa taaaacaagt aataaaatat taatggaaaa
     1981 aataaattcc tgtgtatttg gaacggattc taatcacttt tcttgcccgg agtcattttt
     2041 gacatgcccg ataacgctgg acacacctgc gaatggagtg ttcatgagaa actcacaagg
     2101 tgctgagata tgctctctat atgataagga cacgttagtg caacttgttg aaactggtgg
     2161 agctcatcct ctgagtcgag aacctataac agaatcaatg attatgagaa aagacgaatg
     2221 tcactttgat tcaaaaaaag aatcctttgt tgcaagtgat gcttaaacta gtttaagcat
     2281 cacttgcaac aaagagtgac agaggccgat ccttggcgcc atagtcggcc gcgagaagca
     2341 cacggtcaca ctgcttccgg tagtcaataa cccggtgcac caccagtaga catacgcggc
     2401 tatttaacga ccctgccctg aaccgacgac cgggtcatcg tggccggatc ttgcggcccc
     2461 tcggcttgaa cgaattgtta gacattattt gccgactacc ttggtgatct cgcctttcac
     2521 gtagtggaca aattcttcca actgatctgc gcgcgaggcc aagcgatctt cttcttgtcc
     2581 aagataagcc tgtctagctt caagtatgac gggctgatac tgggccggca ggcgctccat
     2641 tgcccagtcg gcagcgacat ccttcggcgc gattttgccg gttactgcgc tgtaccaaat
     2701 gcgggacaac gtaagcacta catttcgctc atcgccagcc cagtcgggcg gcgagttcca
     2761 tagcgttaag gtttcattta gcgcctcaaa tagatcctgt tcaggaaccg gatcaaagag
     2821 ttcctccgcc gctggaccta ccaaggcaac gctatgttct cttgcttttg tcagcaagat
     2881 agccagatca atgtcgatcg tggctggctc gaagatacct gcaagaatgt cattgcgctg
     2941 ccattctcca aattgcagtt cgcgcttagc tggataacgc cacggaatga tgtcgtcgtg
     3001 cacaacaatg gtgacttcta cagcgcggag aatctcgctc tctccagggg aagccgaagt
     3061 ttccaaaagg tcgttgatca aagctcgccg cgttgtttca tcaagcctta cggtcaccgt
     3121 aaccagcaaa tcaatatcac tgtgtggctt caggccgcca tccactgcgg agccgtacaa
     3181 atgtacggcc agcaacgtcg gttcgagatg gcgctcgatg acgccaacta cctctgatag
     3241 ttgagtcgat acttcggcga tcaccgcttc cctcatactc ttcctttttc aatattattg
     3301 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3361 taaacaaata gctagctcac tcggtcgcta cgctccgggc gtgagactgc ggcgggcgct
     3421 gcggacacat acaaagttac ccacagattc cgtggataag caggggacta acatgtgagg
     3481 caaaacagca gggccgcgcc ggtggcgttt ttccataggc tccgccctcc tgccagagtt
     3541 cacataaaca gacgcttttc cggtgcatct gtgggagccg tgaggctcaa ccatgaatct
     3601 gacagtacgg gcgaaacccg acaggactta aagatcccca ccgtttccgg cgggtcgctc
     3661 cctcttgcgc tctcctgttc cgaccctgcc gtttaccgga tacctgttcc gcctttctcc
     3721 cttacgggaa gtgtggcgct ttctcatagc tcacacactg gtatctcggc tcggtgtagg
     3781 tcgttcgctc caagctgggc tgtaagcaag aactccccgt tcagcccgac tgctgcgcct
     3841 tatccggtaa ctgttcactt gagtccaacc cggaaaagca cggtaaaacg ccactggcag
     3901 cagccattgg taactgggag ttcgcagagg atttgtttag ctaaacacgc ggttgctctt
     3961 gaagtgtgcg ccaaagtccg gctacactgg aaggacagat ttggttgctg tgctctgcga
     4021 aagccagtta ccacggttaa gcagttcccc aactgactta accttcgatc aaaccacctc
     4081 cccaggtggt tttttcgttt acagggcaaa agattacgcg cagaaaaaaa ggatctcaag
     4141 aagatccttt gat
//
