LOCUS       sequence-190657-        3792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the cDHFR-link2-His6-MBP-HRS (258-277) A266Q,A268Q Hs in 
            E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3792)
  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 3792)
  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-Sub64-HRS (258-277) A266Q,A268Q Hs
FEATURES             Location/Qualifiers
     source          1..3792
                     /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(2124..2915)
                     /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(2916..3007)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(3055..3792,1))
                     /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 cggcaggaag aagaggaact
     1861 gcagctccaa ctccagctgt cccagtctga agcggaagaa aaataaacta gttaagtcga
     1921 acagaaagta acgtattgta cacggccgaa gcttggcgcc atagcggccg cgagaagcac
     1981 acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct
     2041 atttaacgac cctgccctga accgacgacc gggtcatcgt ggccggatct tgcggcccct
     2101 cggcttgaac gaattgttag acattatttg ccgactacct tggtgatctc gcctttcacg
     2161 tagtggacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc ttcttgtcca
     2221 agataagcct gtctagcttc aagtatgacg ggctgatact gggccggcag gcgctccatt
     2281 gcccagtcgg cagcgacatc cttcggcgcg attttgccgg ttactgcgct gtaccaaatg
     2341 cgggacaacg taagcactac atttcgctca tcgccagccc agtcgggcgg cgagttccat
     2401 agcgttaagg tttcatttag cgcctcaaat agatcctgtt caggaaccgg atcaaagagt
     2461 tcctccgccg ctggacctac caaggcaacg ctatgttctc ttgcttttgt cagcaagata
     2521 gccagatcaa tgtcgatcgt ggctggctcg aagatacctg caagaatgtc attgcgctgc
     2581 cattctccaa attgcagttc gcgcttagct ggataacgcc acggaatgat gtcgtcgtgc
     2641 acaacaatgg tgacttctac agcgcggaga atctcgctct ctccagggga agccgaagtt
     2701 tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat caagccttac ggtcaccgta
     2761 accagcaaat caatatcact gtgtggcttc aggccgccat ccactgcgga gccgtacaaa
     2821 tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga cgccaactac ctctgatagt
     2881 tgagtcgata cttcggcgat caccgcttcc ctcatactct tcctttttca atattattga
     2941 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3001 aaacaaatag ctagctcact cggtcgctac gctccgggcg tgagactgcg gcgggcgctg
     3061 cggacacata caaagttacc cacagattcc gtggataagc aggggactaa catgtgaggc
     3121 aaaacagcag ggccgcgccg gtggcgtttt tccataggct ccgccctcct gccagagttc
     3181 acataaacag acgcttttcc ggtgcatctg tgggagccgt gaggctcaac catgaatctg
     3241 acagtacggg cgaaacccga caggacttaa agatccccac cgtttccggc gggtcgctcc
     3301 ctcttgcgct ctcctgttcc gaccctgccg tttaccggat acctgttccg cctttctccc
     3361 ttacgggaag tgtggcgctt tctcatagct cacacactgg tatctcggct cggtgtaggt
     3421 cgttcgctcc aagctgggct gtaagcaaga actccccgtt cagcccgact gctgcgcctt
     3481 atccggtaac tgttcacttg agtccaaccc ggaaaagcac ggtaaaacgc cactggcagc
     3541 agccattggt aactgggagt tcgcagagga tttgtttagc taaacacgcg gttgctcttg
     3601 aagtgtgcgc caaagtccgg ctacactgga aggacagatt tggttgctgt gctctgcgaa
     3661 agccagttac cacggttaag cagttcccca actgacttaa ccttcgatca aaccacctcc
     3721 ccaggtggtt ttttcgttta cagggcaaaa gattacgcgc agaaaaaaag gatctcaaga
     3781 agatcctttg at
//
