LOCUS       sequence-465027-        6570 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express BirA tagged to the N-terminus of TRAF6..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6570)
  AUTHORS   Mukhopadhyay U, Levantovsky S, Carusone TM, Gharbi S, Stein F, 
            Behrends C, Bhogaraju S
  TITLE     A ubiquitin-specific, proximity-based labeling approach for the 
            identification of ubiquitin ligase substrates.
  JOURNAL   Sci Adv. 2024 Aug 9;10(32):eadp3000. doi: 10.1126/sciadv.adp3000. 
            Epub 2024 Aug 9.
  PUBMED    39121224
REFERENCE   2  (bases 1 to 6570)
  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: "Sci Adv."; 
            date: "2024-08-9"; volume: "10(32):eadp3000. doi"; pages: " 
            10.1126/sciadv.adp3000. Epub 2024 Aug 9"
COMMENT     Sequence Label: pEHAC1- HA-BirA-Traf6
FEATURES             Location/Qualifiers
     source          1..6570
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     417..436
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(735..754)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    779..826
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1155..1743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1644..1663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1918..2221
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2222..2425
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2376..2396
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2473..2499
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2521..3483
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2521..3483
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     polyA_signal    5215..5336
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5252..5271)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5306..5325
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5343..5798)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5480..5501)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5692..5711
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5825..5929
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5931..6288
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5931..6122
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5954..5974)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6139..6274
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(6323..6570,1..547)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             join(6323..6570,1..547)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6377..6396)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
       61 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
      121 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
      181 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
      241 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
      301 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
      361 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
      421 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
      481 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
      541 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
      601 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
      661 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct
      721 agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
      781 gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt
      841 cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc
      901 cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca
      961 gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt
     1021 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     1081 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1141 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     1201 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     1261 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     1321 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     1381 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     1441 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1501 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     1561 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     1621 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     1681 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     1741 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     1801 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcattag
     1861 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     1921 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     1981 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     2041 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     2101 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     2161 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     2221 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     2281 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     2341 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     2401 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct agcgctaccg
     2461 gtcgccacca tgtacccata cgacgtccca gactacgctt ccggactcag atctcgaggg
     2521 atgaaggata acaccgtgcc actgaaattg attgccctgt tagcgaacgg tgaatttcac
     2581 tctggcgagc agttgggtga aacgctggga atgagccggg cggctattaa taaacacatt
     2641 cagacactgc gtgactgggg cgttgatgtc tttaccgttc cgggtaaagg atacagcctg
     2701 cctgagccta tccagttact taatgctaaa cagatattgg gtcagctgga tggcggtagt
     2761 gtagccgtgc tgcctgtgat tgactccacg aatcagtacc ttcttgatcg tatcggagag
     2821 cttaaatcgg gcgatgcttg cattgcagaa taccagcagg ctggccgtgg tcgccggggt
     2881 cggaaatggt tttcgccttt tggcgcaaac ttatatttgt cgatgttctg gcgtctggaa
     2941 caaggcccgg cggcggcgat tggtttaagt ctggttatcg gtatcgtgat ggcggaagta
     3001 ttacgcaagc tgggtgcaga taaagttcgt gttaaatggc ctaatgacct ctatctgcag
     3061 gatcgcaagc tggcaggcat tctggtggag ctgactggca aaactggcga tgcggcgcaa
     3121 atagtcattg gagccgggat caacatggca atgcgccgtg ttgaagagag tgtcgttaat
     3181 caggggtgga tcacgctgca ggaagcgggg atcaatctcg atcgtaatac gttggcggcc
     3241 atgctaatac gtgaattacg tgctgcgttg gaactcttcg aacaagaagg attggcacct
     3301 tatctgtcgc gctgggaaaa gctggataat tttattaatc gcccagtgaa acttatcatt
     3361 ggtgataaag aaatatttgg catttcacgc ggaatagaca aacagggggc tttattactt
     3421 gagcaggatg gaataataaa accctggatg ggcggtgaaa tatccctgcg tagtgcagaa
     3481 aaacaagctt cgaattctgc agtcgacggt accatgagtc tgctaaactg tgaaaacagc
     3541 tgtggatcca gccagtctga aagtgactgc tgtgtggcca tggccagctc ctgtagcgct
     3601 gtaacaaaag atgatagtgt gggtggaact gccagcacgg ggaacctctc cagctcattt
     3661 atggaggaga tccagggata tgatgtagag tttgacccac ccctggaaag caagtatgaa
     3721 tgccccatct gcttgatggc attacgagaa gcagtgcaaa cgccatgcgg ccataggttc
     3781 tgcaaagcct gcatcataaa atcaataagg gatgcaggtc acaaatgtcc agttgacaat
     3841 gaaatactgc tggaaaatca actatttcca gacaattttg caaaacgtga gattctttct
     3901 ctgatggtga aatgtccaaa tgaaggttgt ttgcacaaga tggaactgag acatcttgag
     3961 gatcatcaag cacattgtga gtttgctctt atggattgtc cccaatgcca gcgtcccttc
     4021 caaaaattcc atattaatat tcacattctg aaggattgtc caaggagaca ggtttcttgt
     4081 gacaactgtg ctgcatcaat ggcatttgaa gataaagaga tccatgacca gaactgtcct
     4141 ttggcaaatg tcatctgtga atactgcaat actatactca tcagagaaca gatgcctaat
     4201 cattatgatc tagactgccc tacagcccca attccatgca cattcagtac ttttggttgc
     4261 catgaaaaga tgcagaggaa tcacttggca cgccacctac aagagaacac ccagtcacac
     4321 atgagaatgt tggcccaggc tgttcatagt ttgagcgtta tacccgactc tgggtatatc
     4381 tcagaggtcc ggaatttcca ggaaactatt caccagttag agggtcgcct tgtaagacaa
     4441 gaccatcaaa tccgggagct gactgctaaa atggaaactc agagtatgta tgtaagtgag
     4501 ctcaaacgaa ccattcgaac ccttgaggac aaagttgctg aaatcgaagc acagcagtgc
     4561 aatggaattt atatttggaa gattggcaac tttggaatgc atttgaaatg tcaagaagag
     4621 gagaaacctg ttgtgattca tagccctgga ttctacactg gcaaacccgg gtacaaactg
     4681 tgcatgcgct tgcaccttca gttaccgact gctcagcgct gtgcaaacta tatatccctt
     4741 tttgtccaca caatgcaagg agaatatgac agccacctcc cttggccctt ccagggtaca
     4801 atacgcctta caattcttga tcagtctgaa gcacctgtaa ggcaaaacca cgaagagata
     4861 atggatgcca aaccagagct gcttgctttc cagcgaccca caatcccacg gaacccaaaa
     4921 ggttttggct atgtaacttt tatgcatctg gaagccctaa gacaaagaac tttcattaag
     4981 gatgacacat tattagtgcg ctgtgaggtc tccacccgct ttgacatggg tagccttcgg
     5041 agggagggtt ttcagccacg aagtactgat gcaggggtat agtgaggatc caccggatct
     5101 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     5161 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     5221 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     5281 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     5341 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     5401 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     5461 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     5521 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     5581 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     5641 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     5701 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5761 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     5821 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5881 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     5941 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     6001 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     6061 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     6121 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     6181 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     6241 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     6301 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     6361 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     6421 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     6481 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     6541 gggcgttcct tgcgcagctg tgctcgacgt
//
