LOCUS       sequence-163470-        3302 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses the cDHFR-link2-His6-MBP-Epn1 (18-157) E42A Dr in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3302)
  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 3302)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-Sub24-Epn1 (18-157) E42A Dr
FEATURES             Location/Qualifiers
     source          1..3302
                     /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     1167..1186
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1167..1185
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1187..1211
                     /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             1320..1364
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1402..1420)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1416..1463
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1634..2425)
                     /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(2426..2517)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(2565..3302,1))
                     /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 cgcggatgtc agaggcggag ataaaggtaa gggaagcaac
      721 ttctaatgat ccatggggtc cctcaagttc tctgatgtca gcgatcgctg acctgacata
      781 caatgtagtg gccttctcag agattatgag catggtctgg aagagactca acgaccatgg
      841 gaaaaactgg agacatgtat ataaagctat gacactgatg gagtatctga taaagacggg
      901 ttcagagcgg gtagcacagc aatgcaggga aaacatctac gccgttcaaa cgcttaaaga
      961 cttccagtac atagacagag acggcaaaga ccagggtgtg aatgtacggg aaaaggccaa
     1021 gcaactggtg actctgctaa aagacgagga gaggctacga gaggagagga tccatgcact
     1081 caagaccaaa gagaagatgg cacagacctg aactagttaa gtcgaacaga aagtaatcgt
     1141 attgtacacg gccgcataat cgaaattaat acgactcact atagggggaa ttgtgagcgg
     1201 ataacaattc cccatcttag tatattagtt aagtataaga aggagatata catatggcag
     1261 atctcaattg gatatcggcc ggccacgcga tcgctgacgt cggtaccctc gagtctggta
     1321 aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt
     1381 aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     1441 aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac acggtcacac
     1501 tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac
     1561 cctgccctga accgacgacc gggtcatcgt ggccggatct tgcggcccct cggcttgaac
     1621 gaattgttag acattatttg ccgactacct tggtgatctc gcctttcacg tagtggacaa
     1681 attcttccaa ctgatctgcg cgcgaggcca agcgatcttc ttcttgtcca agataagcct
     1741 gtctagcttc aagtatgacg ggctgatact gggccggcag gcgctccatt gcccagtcgg
     1801 cagcgacatc cttcggcgcg attttgccgg ttactgcgct gtaccaaatg cgggacaacg
     1861 taagcactac atttcgctca tcgccagccc agtcgggcgg cgagttccat agcgttaagg
     1921 tttcatttag cgcctcaaat agatcctgtt caggaaccgg atcaaagagt tcctccgccg
     1981 ctggacctac caaggcaacg ctatgttctc ttgcttttgt cagcaagata gccagatcaa
     2041 tgtcgatcgt ggctggctcg aagatacctg caagaatgtc attgcgctgc cattctccaa
     2101 attgcagttc gcgcttagct ggataacgcc acggaatgat gtcgtcgtgc acaacaatgg
     2161 tgacttctac agcgcggaga atctcgctct ctccagggga agccgaagtt tccaaaaggt
     2221 cgttgatcaa agctcgccgc gttgtttcat caagccttac ggtcaccgta accagcaaat
     2281 caatatcact gtgtggcttc aggccgccat ccactgcgga gccgtacaaa tgtacggcca
     2341 gcaacgtcgg ttcgagatgg cgctcgatga cgccaactac ctctgatagt tgagtcgata
     2401 cttcggcgat caccgcttcc ctcatactct tcctttttca atattattga agcatttatc
     2461 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2521 ctagctcact cggtcgctac gctccgggcg tgagactgcg gcgggcgctg cggacacata
     2581 caaagttacc cacagattcc gtggataagc aggggactaa catgtgaggc aaaacagcag
     2641 ggccgcgccg gtggcgtttt tccataggct ccgccctcct gccagagttc acataaacag
     2701 acgcttttcc ggtgcatctg tgggagccgt gaggctcaac catgaatctg acagtacggg
     2761 cgaaacccga caggacttaa agatccccac cgtttccggc gggtcgctcc ctcttgcgct
     2821 ctcctgttcc gaccctgccg tttaccggat acctgttccg cctttctccc ttacgggaag
     2881 tgtggcgctt tctcatagct cacacactgg tatctcggct cggtgtaggt cgttcgctcc
     2941 aagctgggct gtaagcaaga actccccgtt cagcccgact gctgcgcctt atccggtaac
     3001 tgttcacttg agtccaaccc ggaaaagcac ggtaaaacgc cactggcagc agccattggt
     3061 aactgggagt tcgcagagga tttgtttagc taaacacgcg gttgctcttg aagtgtgcgc
     3121 caaagtccgg ctacactgga aggacagatt tggttgctgt gctctgcgaa agccagttac
     3181 cacggttaag cagttcccca actgacttaa ccttcgatca aaccacctcc ccaggtggtt
     3241 ttttcgttta cagggcaaaa gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3301 at
//
