LOCUS       sequence-158196-        3299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the cDHFR-link2-His6-MBP-Epn1 (18-157) Dr in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3299)
  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 3299)
  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-Sub22-Epn1 (18-157) Dr
FEATURES             Location/Qualifiers
     source          1..3299
                     /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             656..679
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     1164..1183
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1164..1182
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1184..1208
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             1317..1361
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1399..1417)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1413..1460
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1631..2422)
                     /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(2423..2514)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(2562..3299,1))
                     /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 tcaccccatg gggatcgata cgaccctgga
      661 agttctgttc caggggcccc cgcggtcaga ggcggagata aaggtaaggg aagcaacttc
      721 taatgatcca tggggtccct caagttctct gatgtcagag atcgctgacc tgacatacaa
      781 tgtagtggcc ttctcagaga ttatgagcat ggtctggaag agactcaacg accatgggaa
      841 aaactggaga catgtatata aagctatgac actgatggag tatctgataa agacgggttc
      901 agagcgggta gcacagcaat gcagggaaaa catctacgcc gttcaaacgc ttaaagactt
      961 ccagtacata gacagagacg gcaaagacca gggtgtgaat gtacgggaaa aggccaagca
     1021 actggtgact ctgctaaaag acgaggagag gctacgagag gagaggatcc atgcactcaa
     1081 gaccaaagag aagatggcac agacctgaac tagttaagtc gaacagaaag taatcgtatt
     1141 gtacacggcc gcataatcga aattaatacg actcactata gggggaattg tgagcggata
     1201 acaattcccc atcttagtat attagttaag tataagaagg agatatacat atggcagatc
     1261 tcaattggat atcggccggc cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag
     1321 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
     1381 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1441 gggtcttgag gggttttttg ctgaaacctc aggcatttga gaagcacacg gtcacactgc
     1501 ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct
     1561 gccctgaacc gacgaccggg tcatcgtggc cggatcttgc ggcccctcgg cttgaacgaa
     1621 ttgttagaca ttatttgccg actaccttgg tgatctcgcc tttcacgtag tggacaaatt
     1681 cttccaactg atctgcgcgc gaggccaagc gatcttcttc ttgtccaaga taagcctgtc
     1741 tagcttcaag tatgacgggc tgatactggg ccggcaggcg ctccattgcc cagtcggcag
     1801 cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa
     1861 gcactacatt tcgctcatcg ccagcccagt cgggcggcga gttccatagc gttaaggttt
     1921 catttagcgc ctcaaataga tcctgttcag gaaccggatc aaagagttcc tccgccgctg
     1981 gacctaccaa ggcaacgcta tgttctcttg cttttgtcag caagatagcc agatcaatgt
     2041 cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt gcgctgccat tctccaaatt
     2101 gcagttcgcg cttagctgga taacgccacg gaatgatgtc gtcgtgcaca acaatggtga
     2161 cttctacagc gcggagaatc tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt
     2221 tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc agcaaatcaa
     2281 tatcactgtg tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt acggccagca
     2341 acgtcggttc gagatggcgc tcgatgacgc caactacctc tgatagttga gtcgatactt
     2401 cggcgatcac cgcttccctc atactcttcc tttttcaata ttattgaagc atttatcagg
     2461 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagcta
     2521 gctcactcgg tcgctacgct ccgggcgtga gactgcggcg ggcgctgcgg acacatacaa
     2581 agttacccac agattccgtg gataagcagg ggactaacat gtgaggcaaa acagcagggc
     2641 cgcgccggtg gcgtttttcc ataggctccg ccctcctgcc agagttcaca taaacagacg
     2701 cttttccggt gcatctgtgg gagccgtgag gctcaaccat gaatctgaca gtacgggcga
     2761 aacccgacag gacttaaaga tccccaccgt ttccggcggg tcgctccctc ttgcgctctc
     2821 ctgttccgac cctgccgttt accggatacc tgttccgcct ttctccctta cgggaagtgt
     2881 ggcgctttct catagctcac acactggtat ctcggctcgg tgtaggtcgt tcgctccaag
     2941 ctgggctgta agcaagaact ccccgttcag cccgactgct gcgccttatc cggtaactgt
     3001 tcacttgagt ccaacccgga aaagcacggt aaaacgccac tggcagcagc cattggtaac
     3061 tgggagttcg cagaggattt gtttagctaa acacgcggtt gctcttgaag tgtgcgccaa
     3121 agtccggcta cactggaagg acagatttgg ttgctgtgct ctgcgaaagc cagttaccac
     3181 ggttaagcag ttccccaact gacttaacct tcgatcaaac cacctcccca ggtggttttt
     3241 tcgtttacag ggcaaaagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgat
//
