LOCUS       sequence-449285-        5020 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses A3-Ub for E3 substrate identification by E-STUB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5020)
  AUTHORS   Huang HT, Lumpkin RJ, Tsai RW, Su S, Zhao X, Xiong Y, Chen J, Mageed
            N, Donovan KA, Fischer ES, Sellers WR
  TITLE     Ubiquitin-specific proximity labeling for the identification of E3 
            ligase substrates.
  JOURNAL   Nat Chem Biol. 2024 Mar 21. doi: 10.1038/s41589-024-01590-9.
  PUBMED    38514884
REFERENCE   2  (bases 1 to 5020)
  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"; journalName: "Nat Chem 
            Biol. 2024 Mar 21. doi: 10.1038/s41589-024-01590-9."
COMMENT     Sequence Label: pRK5-A3-HA-ubiquitin
FEATURES             Location/Qualifiers
     source          1..5020
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      950..959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1010..1036
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1043..1270
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1043..1270
                     /codon_start=1
                     /gene="Homo sapiens UBC"
                     /product="human ubiquitin C"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     regulatory      1307..1316
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1320..1454
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1357..1376)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1411..1430
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1523..1880
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1523..1714
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1546..1566)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1731..1866
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1900..1917)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1900..1916)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1909..1931)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2129..2584
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2216..2235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2426..2447
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2601..2620)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2720..2742
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2780..2798)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2866..2970
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2971..3039,3040..3831)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3189..3208)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4002..4590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4491..4510
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4744..4761
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4878..4899
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4914..4919,4920..4937,4938..4944)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4952..4968
                     /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)."
     primer_bind     4957..4979
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4976..4992
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4976..4992
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgattgaatt cccgaaccga cagtcggtct cttcaccaag gccattcgcg ccaccatggg
      961 actgaacgac atcttcgagg ctcagaaaat cgaagggagc ggaggttcat atccgtacga
     1021 tgtgccggac tatgcgggat ccatgcagat cttcgtgaag accctgactg gtaagaccat
     1081 cactctcgaa gtggagccga gtgacaccat tgagaatgtc aaggcaaaga tccaagacaa
     1141 ggaaggcatc cctcctgacc agcagaggtt gatctttgct gggaaacagc tggaagatgg
     1201 acgcaccctg tctgactaca acatccagaa agagtccacc ctgcacctgg tcctccgtct
     1261 cagaggtggt tgagcggccg ctaagtaagt aaggatcccc agcttggccg ccatggccca
     1321 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     1381 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     1441 atcatgtctg gatcgggaat taattcggcg cagcaccatg gcctgaaata acctctgaaa
     1501 gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     1561 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     1621 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     1681 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     1741 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     1801 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     1861 gaggcctagg cttttgcaaa aagctgttaa cagcttggca ctggccgtcg ttttacaacg
     1921 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     1981 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2041 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2101 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     2161 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2221 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2281 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2341 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2401 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2461 cctatctcgg gctattcttt taatttataa gggattttgc cgatttcggc ctattggtta
     2521 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     2581 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     2641 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2701 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     2761 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     2821 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2881 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2941 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3001 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3061 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3121 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3181 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3241 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3301 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3361 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3421 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3481 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3541 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3601 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3661 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     3721 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3781 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     3841 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     3901 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     3961 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4021 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4081 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4141 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4201 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4261 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4321 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4381 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4441 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4501 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4561 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4621 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4681 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4741 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     4801 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     4861 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     4921 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     4981 aacagctatg acatgattac gaattaattc gagctcgccc
//
