LOCUS       sequence-158229-        3979 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the cDHFR-link2-His6-MBP-Sem1 Sc in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3979)
  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 3979)
  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-Sub100-Sem1 Sc
FEATURES             Location/Qualifiers
     source          1..3979
                     /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             2071..2082
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(2311..3102)
                     /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(3103..3194)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(3242..3979,1))
                     /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 cggatgagta cggacgttgc
     1861 tgctgcacaa gcccaaagta aaatagattt aacaaaaaaa aaaaatgagg aaataaataa
     1921 gaagtctcta gaggaagacg atgaatttga agatttcccc attgacacat gggccaatgg
     1981 tgaaacaata aaaagcaatg ctgtaacgca aacaaacata tgggaagaaa actgggatga
     2041 tgttgaagtt gacgacgatt tcaccaatga attgaaggcc gaattggata gatacaaacg
     2101 ggaaaatcag tgaactagtt aagtcgaaca gaaagtaatc gtattgtaca cggccgcaga
     2161 gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata
     2221 agcggctatt taacgaccct gccctgaacc gacgaccggg tcatcgtggc cggatcttgc
     2281 ggcccctcgg cttgaacgaa ttgttagaca ttatttgccg actaccttgg tgatctcgcc
     2341 tttcacgtag tggacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttc
     2401 ttgtccaaga taagcctgtc tagcttcaag tatgacgggc tgatactggg ccggcaggcg
     2461 ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta
     2521 ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga
     2581 gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc
     2641 aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag
     2701 caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt
     2761 gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc
     2821 gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc
     2881 cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt
     2941 caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc
     3001 gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc
     3061 tgatagttga gtcgatactt cggcgatcac cgcttccctc atactcttcc tttttcaata
     3121 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3181 gaaaaataaa caaatagcta gctcactcgg tcgctacgct ccgggcgtga gactgcggcg
     3241 ggcgctgcgg acacatacaa agttacccac agattccgtg gataagcagg ggactaacat
     3301 gtgaggcaaa acagcagggc cgcgccggtg gcgtttttcc ataggctccg ccctcctgcc
     3361 agagttcaca taaacagacg cttttccggt gcatctgtgg gagccgtgag gctcaaccat
     3421 gaatctgaca gtacgggcga aacccgacag gacttaaaga tccccaccgt ttccggcggg
     3481 tcgctccctc ttgcgctctc ctgttccgac cctgccgttt accggatacc tgttccgcct
     3541 ttctccctta cgggaagtgt ggcgctttct catagctcac acactggtat ctcggctcgg
     3601 tgtaggtcgt tcgctccaag ctgggctgta agcaagaact ccccgttcag cccgactgct
     3661 gcgccttatc cggtaactgt tcacttgagt ccaacccgga aaagcacggt aaaacgccac
     3721 tggcagcagc cattggtaac tgggagttcg cagaggattt gtttagctaa acacgcggtt
     3781 gctcttgaag tgtgcgccaa agtccggcta cactggaagg acagatttgg ttgctgtgct
     3841 ctgcgaaagc cagttaccac ggttaagcag ttccccaact gacttaacct tcgatcaaac
     3901 cacctcccca ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga aaaaaaggat
     3961 ctcaagaaga tcctttgat
//
