LOCUS       sequence-158200-        6659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the nDHFR-link1-His6-Ub, UBC1 Sc and UBA1 Ta in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6659)
  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 6659)
  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-Ub01-UBC1 Sc
FEATURES             Location/Qualifiers
     source          1..6659
                     /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             4983..4994
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     rep_origin      complement(5995..6583)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6075..6094)
                     /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 atgtctaggg ctaagagaat tatgaaagaa
     2041 atccaagctg tgaaggatga tcctgcagct cacattactc ttgaatttgt gagtgaatct
     2101 gatatccacc atttaaaagg cacatttttg ggcccacctg gaacacctta cgagggtggc
     2161 aaatttgtcg tggatatcga agtacctatg gagtatccat tcaaaccacc aaagatgcag
     2221 ttcgacacaa aagtatacca tccaaatata tcatcagtga caggtgccat ttgtttagat
     2281 attcttaaga atgcatggtc gccagtgata acactaaagt ctgcattgat ttcgctacag
     2341 gcgctactac aatctccaga gcctaacgat cctcaagatg cagaagtagc acaacactat
     2401 ttacgggata gagaatcttt taataagact gctgcactat ggacgaggtt atacgccagt
     2461 gaaacttcca atggtcaaaa aggcaatgta gaggagtctg atttatatgg gattgaccac
     2521 gatctgattg acgagtttga atctcaaggt tttgaaaagg acaagattgt ggaagtgttg
     2581 agacgattag gcgtcaagtc cttagacccc aatgacaaca acacagccaa ccgtatcatc
     2641 gaggaattgt tgaagtgagg cgcgccacaa ttccgacagg aaacagctat gcttccccgg
     2701 aagcgggaga tcgtcgccgg cgaggtcgag gacttgcaga agaagacccg cgccggggag
     2761 ggggaggtca ctagggagga aggagacgca gccatggcag ggagggggaa cgagatcgac
     2821 gaggacctgc acagccgcca gctcgccgtc tatgggcgcg agaccatgaa gcgcctcttc
     2881 ggctccaacg tcctcgtctc tggactccag ggtctcggtg ctgagatcgc aaagaacctt
     2941 gtccttgcgg gtgtcaagtc tgtaaccttg catgatgatg gtaacgtgga gctgtgggac
     3001 ttatcaagca acttcttcct ctcggagaat gatgttgggc aaaaccgtgc tcaagcttgt
     3061 gtacagaagc tacaagagct gaacaatgct gtcctcgtct ctgccttaac cggtgatttg
     3121 accaaggagc acctttctaa attccaggcc gttgtcttca ctgatatcag cttagataaa
     3181 gcaattgaat ttgatgatta ctgccacagc caacagccac ccattgcgtt catcaagtct
     3241 gaagttcgtg gtctttttgg cagtgttttt tgtgactttg gtcctgaatt tactgttttg
     3301 gatgtggatg gagaggaacc acatacagga attgttgcat caatcagcaa tgacaatcca
     3361 gcacttgtat cttgtgtgga cgatgagcgt ctggagttcc aggatggtga tctagttgtt
     3421 ttctctgaag tccatggaat gactgagctg aatgatggaa aaccaagaaa agttaagaat
     3481 gcaaggcctt actctttctt cttggaagaa gacacttcct catttggcgc atatgttaga
     3541 ggtggtattg tcacacaggt caagccaccg aaggttatta aattcaaacc cttgaaagag
     3601 gccatgtcag agccgggaga atttctcatg agtgatttct ccaaatttga gcggccacct
     3661 cttctgcatt tggcattcca agctttggat aagtttagga ctgagttgag cagattccct
     3721 gttgctgggt ccactgatga tgtgcaaagg gtgatagagt atgctattag cattaatgat
     3781 actctgggag ataggaaact tgaagaaatt gacaaaaagc tcctgcatca ttttgccagt
     3841 ggttccaggg ctgttctgaa tcctatggct gcgatgtttg gtggtattgt gggtcaggag
     3901 gtagtgaaag cttgctcagg gaaatttcat ccactttatc agttcttcta ttttgattct
     3961 gtcgagtctc tccctgttga ccccttggag cctggtgatt tgaagccgaa gaacagtaga
     4021 tatgatgctc aaatcagcgt atttggatct aagcttcaaa acaaactgga ggaagcaaaa
     4081 atctttatgg tcggttctgg ggcacttgga tgtgagttct tgaagaacct ggcactaatg
     4141 ggtatttctt gcagccagaa tggaaatctg actctgacag atgatgatgt cattgaaaag
     4201 agtaatctga gccgccaatt tcttttccgt gactggaaca ttgggcaacc taagtccaca
     4261 gttgctgcta cagctgctat ggtaattaat cctaaacttc atgtcgaggc ccttcagaac
     4321 agagcaagtc ctgagactga aaatgtgttt aatgatgcct tctgggagaa ccttgatgct
     4381 gttgtcaatg cccttgacaa tgttactgca agaatgtaca tagactccag atgtgtatat
     4441 ttccagaagc cacttttgga atctgggact ctgggtgcta aatgcaatac acagatggtc
     4501 atccctcacc taacagagaa ctatggggct tcaagggatc cacctgaaaa gcaggcacct
     4561 atgtgcactg tacattcatt tcctcataac attgatcatt gcctaacctg ggctaggtct
     4621 gagtttgagg gtttacttga gaagactccc actgaagtaa atgctttcct gtcaaatcct
     4681 actacataca taagtgcagc aagaactgct ggtgatgcac aggctagaga tcagcttgaa
     4741 cgtgttattg agtgtcttga cagagacaag tgcgagacgt tccaggattc tattacctgg
     4801 gcccggctta agtttgagga ttacttctcc aaccgtgtga agcagctgac gttcactttc
     4861 ccagaagact cgatgaccag ctctggtgct cctttctggt ctgctccaaa gcggttccca
     4921 cgacctgtgg agttctcgtc cagtgatcag agtcagctta gctttatttt ggctgctgca
     4981 atattgaggg cagagacttt tggaataccc atacctgagt gggccaaaac cccaaacaag
     5041 ctggctgctg aagctgtcga caaggtgatt gtcccggatt ttcaaccaaa gcagggggtt
     5101 aagatagtta cacatgagaa ggccactagt ctgtcctctg catctgttga cgacgctgct
     5161 gtcattgaag agcttattgc caagttagaa gaagtttcca aaacactgcc atcagggttc
     5221 cacatgaacc caatacagtt tgagaaggat gatgacacaa acttccatat ggatgtgata
     5281 gctggttttg ccaacatgag ggcaaggaac tacagcattc ctgaagtgga caagctcaag
     5341 gccaagttta tagccggcag gatcatccca gctatcgcca cctccaccgc gatggccact
     5401 ggcctcgtct gccttgagct ttacaaagcc cttgctggtg gacacaaggt tgaagactac
     5461 cgcaacacgt ttgcaaacct cgctatccct ctcttctcca ttgctgaacc agttccaccc
     5521 aagaccatca agcaccagga gttgtcgtgg acggtctggg accggtggac tgtgacgggc
     5581 aacatcacgc tgagggagct cctggagtgg ctcaaggaga agggcctgaa cgcttacagc
     5641 atatcctgtg gcacctcgct gctgtacaac tccatgttcc ccaggcacaa ggaacggctt
     5701 gacaggaagg tagttgatgt tgccagggag gtggccaaga tggaggtgcc ctcttaccgg
     5761 cgtcatctcg acgtcgtggt ggcgtgtgag gatgacgacg acaatgatgt cgacatccca
     5821 cttgtgtcgg tctacttccg ctgacctagg cgttcggctg cggcgagcgg tatcagctca
     5881 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5941 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     6001 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     6061 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6121 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6181 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6241 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6301 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6361 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6421 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6481 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6541 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6601 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatga
//
