LOCUS       sequence-163949-        3362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the nDHFR-link1-His6-Ub and UBC4 Sc in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3362)
  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 3362)
  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: pND-Ub20-deltaE1
FEATURES             Location/Qualifiers
     source          1..3362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(12..106)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(137..931)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(248..267)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     858..877
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1110..1128
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1149..1167,1168..1173,1174..1190,1191..1196,
                     1197..1222)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1149..1167
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1174..1192
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1232..1243
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1688..1705
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1727..1747
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1760..1987
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     rep_origin      complement(2698..3286)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2778..2797)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa
       61 tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag cgagctctcg
      121 aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
      181 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
      241 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
      301 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
      361 catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga
      421 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
      481 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
      541 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
      601 cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc
      661 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
      721 ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg
      781 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
      841 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
      901 aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat
      961 cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt
     1021 tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccgac gtctaagaaa
     1081 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     1141 acctcgagtc cctatcagtg atagagattg acatccctat cagtgataga gatactgagc
     1201 acatcagcag gacgcactga ccgaattcat taaagaggag aaaggtacca tggttcgacc
     1261 attgaactgc atcgtcgccg tgtcccaaaa tatggggatt ggcaagaacg gagacctacc
     1321 ctggcctccg ctcaggaacg agttcaagta cttccaaaga atgaccacaa cctcttcagt
     1381 ggaaggtaaa cagaatctgg tgattatggg taggaaaacc tggttctcca ttcctgagaa
     1441 gaatcgacct ttaaaggaca gaattaatat agttctcagt agagaactca aagaaccacc
     1501 acgaggagct cattttcttg ccaaaagttt ggatgatgcc ttaagactta ttgaacaacc
     1561 ggaattggca ttaattaagg cggctcagcg ggctcgggaa gcggagcggg atctggcagc
     1621 ggcggtagcg caggcagcag cgggtcaagc ggtgcctcga gcggcgcggc cgcaaatgtc
     1681 gtactaccat caccatcacc atcacgatta cgatatccca acgaccgaaa acctgtattt
     1741 tcagggcgcc atgggatcca tgcagatctt cgtgaagact ctgactggta agaccatcac
     1801 cctcgaggtg gagcccagtg acaccatcga gaatgtcaag gcaaagatcc aagataagga
     1861 aggcattcct cctgatcagc agaggttgat ctttgccgga aaacagctgg aagatggtcg
     1921 taccctgtct gactacaaca tccagaaaga gtccaccttg cacctggtac tccgtctcag
     1981 aggtgggtga gaattctaag gaaatccatt atgtcttctt ctaaacgtat tgctaaagaa
     2041 ctaagtgatc tagaaagaga tccacctact tcatgttcag ccggtccagt cggcgatgat
     2101 ctatatcact ggcaagcatc catcatggga cctgccgatt ccccatatgc cggcggtgtt
     2161 ttcttcttgt ctatccattt cccaaccgac tacccattca agccaccaaa gatctccttc
     2221 acaaccaaga tatatcatcc aaatatcaat gccaatggta acatctgtct ggacatccta
     2281 aaggatcaat ggtctccagc tctaactcta tcgaaggtcc tattatccat ctgttctttg
     2341 ttaacagacg ctaatcctga cgatccttta gtaccagaaa tcgctcatat ctacaagact
     2401 gacagaccca agtacgaagc tacagccaga gaatggacaa agaaatacgc tgtataaggc
     2461 gcgccacaat tccgacagga aacagctatg cttccccgga agcgggagat cgtcgccggc
     2521 ccacttgtgt cggtctactt ccgctgacct aggcgttcgg ctgcggcgag cggtatcagc
     2581 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2641 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2701 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2761 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2821 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2881 ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2941 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3001 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3061 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3121 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3181 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3241 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3301 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3361 ga
//
