LOCUS       sequence-165372-        6478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the nDHFR-link1-His6-Ub, UBCH5B Hs and UBA1Ta in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6478)
  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 6478)
  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-Ub16-UBCH5B Hs
FEATURES             Location/Qualifiers
     source          1..6478
                     /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             4802..4813
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     rep_origin      complement(5814..6402)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5894..5913)
                     /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 gaattcgccc ttcggggtac ccttaaggaa atccattatg gctctgaaga
     2041 gaatccacaa ggaattgaat gatctggcac gggaccctcc agcacagtgt tcagcaggtc
     2101 ctgttggaga tgatatgttc cattggcaag ctacaataat ggggccaaat gacagtccct
     2161 atcagggtgg agtatttttc ttgacaattc atttcccaac agattacccc ttcaaaccac
     2221 ctaaggttgc atttacaaca agaatttatc atccaaatat taacagtaat ggcagcattt
     2281 gtcttgatat tctacgatca cagtggtctc cagcactaac tatttcaaaa gtactcttgt
     2341 ccatctgttc tctgttgtgt gatcccaatc cagatgatcc tttagtacct gagattgctc
     2401 ggatctacaa aacagataga gaaaagtaca acagaatagc tcgggaatgg actcagaagt
     2461 atgcgatgta aggtaccccg aagggcgaat tccgacagga aacagctatg cttccccgga
     2521 agcgggagat cgtcgccggc gaggtcgagg acttgcagaa gaagacccgc gccggggagg
     2581 gggaggtcac tagggaggaa ggagacgcag ccatggcagg gagggggaac gagatcgacg
     2641 aggacctgca cagccgccag ctcgccgtct atgggcgcga gaccatgaag cgcctcttcg
     2701 gctccaacgt cctcgtctct ggactccagg gtctcggtgc tgagatcgca aagaaccttg
     2761 tccttgcggg tgtcaagtct gtaaccttgc atgatgatgg taacgtggag ctgtgggact
     2821 tatcaagcaa cttcttcctc tcggagaatg atgttgggca aaaccgtgct caagcttgtg
     2881 tacagaagct acaagagctg aacaatgctg tcctcgtctc tgccttaacc ggtgatttga
     2941 ccaaggagca cctttctaaa ttccaggccg ttgtcttcac tgatatcagc ttagataaag
     3001 caattgaatt tgatgattac tgccacagcc aacagccacc cattgcgttc atcaagtctg
     3061 aagttcgtgg tctttttggc agtgtttttt gtgactttgg tcctgaattt actgttttgg
     3121 atgtggatgg agaggaacca catacaggaa ttgttgcatc aatcagcaat gacaatccag
     3181 cacttgtatc ttgtgtggac gatgagcgtc tggagttcca ggatggtgat ctagttgttt
     3241 tctctgaagt ccatggaatg actgagctga atgatggaaa accaagaaaa gttaagaatg
     3301 caaggcctta ctctttcttc ttggaagaag acacttcctc atttggcgca tatgttagag
     3361 gtggtattgt cacacaggtc aagccaccga aggttattaa attcaaaccc ttgaaagagg
     3421 ccatgtcaga gccgggagaa tttctcatga gtgatttctc caaatttgag cggccacctc
     3481 ttctgcattt ggcattccaa gctttggata agtttaggac tgagttgagc agattccctg
     3541 ttgctgggtc cactgatgat gtgcaaaggg tgatagagta tgctattagc attaatgata
     3601 ctctgggaga taggaaactt gaagaaattg acaaaaagct cctgcatcat tttgccagtg
     3661 gttccagggc tgttctgaat cctatggctg cgatgtttgg tggtattgtg ggtcaggagg
     3721 tagtgaaagc ttgctcaggg aaatttcatc cactttatca gttcttctat tttgattctg
     3781 tcgagtctct ccctgttgac cccttggagc ctggtgattt gaagccgaag aacagtagat
     3841 atgatgctca aatcagcgta tttggatcta agcttcaaaa caaactggag gaagcaaaaa
     3901 tctttatggt cggttctggg gcacttggat gtgagttctt gaagaacctg gcactaatgg
     3961 gtatttcttg cagccagaat ggaaatctga ctctgacaga tgatgatgtc attgaaaaga
     4021 gtaatctgag ccgccaattt cttttccgtg actggaacat tgggcaacct aagtccacag
     4081 ttgctgctac agctgctatg gtaattaatc ctaaacttca tgtcgaggcc cttcagaaca
     4141 gagcaagtcc tgagactgaa aatgtgttta atgatgcctt ctgggagaac cttgatgctg
     4201 ttgtcaatgc ccttgacaat gttactgcaa gaatgtacat agactccaga tgtgtatatt
     4261 tccagaagcc acttttggaa tctgggactc tgggtgctaa atgcaataca cagatggtca
     4321 tccctcacct aacagagaac tatggggctt caagggatcc acctgaaaag caggcaccta
     4381 tgtgcactgt acattcattt cctcataaca ttgatcattg cctaacctgg gctaggtctg
     4441 agtttgaggg tttacttgag aagactccca ctgaagtaaa tgctttcctg tcaaatccta
     4501 ctacatacat aagtgcagca agaactgctg gtgatgcaca ggctagagat cagcttgaac
     4561 gtgttattga gtgtcttgac agagacaagt gcgagacgtt ccaggattct attacctggg
     4621 cccggcttaa gtttgaggat tacttctcca accgtgtgaa gcagctgacg ttcactttcc
     4681 cagaagactc gatgaccagc tctggtgctc ctttctggtc tgctccaaag cggttcccac
     4741 gacctgtgga gttctcgtcc agtgatcaga gtcagcttag ctttattttg gctgctgcaa
     4801 tattgagggc agagactttt ggaataccca tacctgagtg ggccaaaacc ccaaacaagc
     4861 tggctgctga agctgtcgac aaggtgattg tcccggattt tcaaccaaag cagggggtta
     4921 agatagttac acatgagaag gccactagtc tgtcctctgc atctgttgac gacgctgctg
     4981 tcattgaaga gcttattgcc aagttagaag aagtttccaa aacactgcca tcagggttcc
     5041 acatgaaccc aatacagttt gagaaggatg atgacacaaa cttccatatg gatgtgatag
     5101 ctggttttgc caacatgagg gcaaggaact acagcattcc tgaagtggac aagctcaagg
     5161 ccaagtttat agccggcagg atcatcccag ctatcgccac ctccaccgcg atggccactg
     5221 gcctcgtctg ccttgagctt tacaaagccc ttgctggtgg acacaaggtt gaagactacc
     5281 gcaacacgtt tgcaaacctc gctatccctc tcttctccat tgctgaacca gttccaccca
     5341 agaccatcaa gcaccaggag ttgtcgtgga cggtctggga ccggtggact gtgacgggca
     5401 acatcacgct gagggagctc ctggagtggc tcaaggagaa gggcctgaac gcttacagca
     5461 tatcctgtgg cacctcgctg ctgtacaact ccatgttccc caggcacaag gaacggcttg
     5521 acaggaaggt agttgatgtt gccagggagg tggccaagat ggaggtgccc tcttaccggc
     5581 gtcatctcga cgtcgtggtg gcgtgtgagg atgacgacga caatgatgtc gacatcccac
     5641 ttgtgtcggt ctacttccgc tgacctaggc gttcggctgc ggcgagcggt atcagctcac
     5701 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5761 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5821 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5881 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5941 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6001 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6061 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6121 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6181 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6241 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6301 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6361 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6421 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatga
//
