LOCUS       sequence-165713-        6004 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the nDHFR-link1-His6-Ub and UBA1Ta in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6004)
  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 6004)
  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-Ub17-deltaE2
FEATURES             Location/Qualifiers
     source          1..6004
                     /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"
     CDS             4328..4339
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     rep_origin      complement(5340..5928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5420..5439)
                     /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 actagttcgg gggggcgcgc cggtctagag acaggaaaca gctatgcttc
     2041 cccggaagcg ggagatcgtc gccggcgagg tcgaggactt gcagaagaag acccgcgccg
     2101 gggaggggga ggtcactagg gaggaaggag acgcagccat ggcagggagg gggaacgaga
     2161 tcgacgagga cctgcacagc cgccagctcg ccgtctatgg gcgcgagacc atgaagcgcc
     2221 tcttcggctc caacgtcctc gtctctggac tccagggtct cggtgctgag atcgcaaaga
     2281 accttgtcct tgcgggtgtc aagtctgtaa ccttgcatga tgatggtaac gtggagctgt
     2341 gggacttatc aagcaacttc ttcctctcgg agaatgatgt tgggcaaaac cgtgctcaag
     2401 cttgtgtaca gaagctacaa gagctgaaca atgctgtcct cgtctctgcc ttaaccggtg
     2461 atttgaccaa ggagcacctt tctaaattcc aggccgttgt cttcactgat atcagcttag
     2521 ataaagcaat tgaatttgat gattactgcc acagccaaca gccacccatt gcgttcatca
     2581 agtctgaagt tcgtggtctt tttggcagtg ttttttgtga ctttggtcct gaatttactg
     2641 ttttggatgt ggatggagag gaaccacata caggaattgt tgcatcaatc agcaatgaca
     2701 atccagcact tgtatcttgt gtggacgatg agcgtctgga gttccaggat ggtgatctag
     2761 ttgttttctc tgaagtccat ggaatgactg agctgaatga tggaaaacca agaaaagtta
     2821 agaatgcaag gccttactct ttcttcttgg aagaagacac ttcctcattt ggcgcatatg
     2881 ttagaggtgg tattgtcaca caggtcaagc caccgaaggt tattaaattc aaacccttga
     2941 aagaggccat gtcagagccg ggagaatttc tcatgagtga tttctccaaa tttgagcggc
     3001 cacctcttct gcatttggca ttccaagctt tggataagtt taggactgag ttgagcagat
     3061 tccctgttgc tgggtccact gatgatgtgc aaagggtgat agagtatgct attagcatta
     3121 atgatactct gggagatagg aaacttgaag aaattgacaa aaagctcctg catcattttg
     3181 ccagtggttc cagggctgtt ctgaatccta tggctgcgat gtttggtggt attgtgggtc
     3241 aggaggtagt gaaagcttgc tcagggaaat ttcatccact ttatcagttc ttctattttg
     3301 attctgtcga gtctctccct gttgacccct tggagcctgg tgatttgaag ccgaagaaca
     3361 gtagatatga tgctcaaatc agcgtatttg gatctaagct tcaaaacaaa ctggaggaag
     3421 caaaaatctt tatggtcggt tctggggcac ttggatgtga gttcttgaag aacctggcac
     3481 taatgggtat ttcttgcagc cagaatggaa atctgactct gacagatgat gatgtcattg
     3541 aaaagagtaa tctgagccgc caatttcttt tccgtgactg gaacattggg caacctaagt
     3601 ccacagttgc tgctacagct gctatggtaa ttaatcctaa acttcatgtc gaggcccttc
     3661 agaacagagc aagtcctgag actgaaaatg tgtttaatga tgccttctgg gagaaccttg
     3721 atgctgttgt caatgccctt gacaatgtta ctgcaagaat gtacatagac tccagatgtg
     3781 tatatttcca gaagccactt ttggaatctg ggactctggg tgctaaatgc aatacacaga
     3841 tggtcatccc tcacctaaca gagaactatg gggcttcaag ggatccacct gaaaagcagg
     3901 cacctatgtg cactgtacat tcatttcctc ataacattga tcattgccta acctgggcta
     3961 ggtctgagtt tgagggttta cttgagaaga ctcccactga agtaaatgct ttcctgtcaa
     4021 atcctactac atacataagt gcagcaagaa ctgctggtga tgcacaggct agagatcagc
     4081 ttgaacgtgt tattgagtgt cttgacagag acaagtgcga gacgttccag gattctatta
     4141 cctgggcccg gcttaagttt gaggattact tctccaaccg tgtgaagcag ctgacgttca
     4201 ctttcccaga agactcgatg accagctctg gtgctccttt ctggtctgct ccaaagcggt
     4261 tcccacgacc tgtggagttc tcgtccagtg atcagagtca gcttagcttt attttggctg
     4321 ctgcaatatt gagggcagag acttttggaa tacccatacc tgagtgggcc aaaaccccaa
     4381 acaagctggc tgctgaagct gtcgacaagg tgattgtccc ggattttcaa ccaaagcagg
     4441 gggttaagat agttacacat gagaaggcca ctagtctgtc ctctgcatct gttgacgacg
     4501 ctgctgtcat tgaagagctt attgccaagt tagaagaagt ttccaaaaca ctgccatcag
     4561 ggttccacat gaacccaata cagtttgaga aggatgatga cacaaacttc catatggatg
     4621 tgatagctgg ttttgccaac atgagggcaa ggaactacag cattcctgaa gtggacaagc
     4681 tcaaggccaa gtttatagcc ggcaggatca tcccagctat cgccacctcc accgcgatgg
     4741 ccactggcct cgtctgcctt gagctttaca aagcccttgc tggtggacac aaggttgaag
     4801 actaccgcaa cacgtttgca aacctcgcta tccctctctt ctccattgct gaaccagttc
     4861 cacccaagac catcaagcac caggagttgt cgtggacggt ctgggaccgg tggactgtga
     4921 cgggcaacat cacgctgagg gagctcctgg agtggctcaa ggagaagggc ctgaacgctt
     4981 acagcatatc ctgtggcacc tcgctgctgt acaactccat gttccccagg cacaaggaac
     5041 ggcttgacag gaaggtagtt gatgttgcca gggaggtggc caagatggag gtgccctctt
     5101 accggcgtca tctcgacgtc gtggtggcgt gtgaggatga cgacgacaat gatgtcgaca
     5161 tcccacttgt gtcggtctac ttccgctgac ctaggcgttc ggctgcggcg agcggtatca
     5221 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5281 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5341 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5401 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5461 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5521 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5581 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5641 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5701 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5761 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5821 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5881 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5941 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6001 atga
//
