LOCUS       sequence-464921-        6582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express BirA tagged to the N-terminus of TRAF6 with a GSGS linker 
            between BirA and TRAF6..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6582)
  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 6582)
  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-GSGS-Traf6
FEATURES             Location/Qualifiers
     source          1..6582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(478..1066)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(558..577)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    1395..1442
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     1467..1486
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(1674..2468)
                     /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             complement(1674..2468)
                     /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(1785..1804)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2395..2414
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2503..2860)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2517..2652
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        2669..2860
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     2817..2837
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(2862..2966)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2993..3448
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3080..3099)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3290..3311
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    3455..3576
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3466..3485)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3520..3539
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5320..6282)
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             complement(5320..6282)
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             complement(6304..6330)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(6378..6581)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6407..6427)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        join(6582,1..303)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
       61 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
      121 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
      181 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
      241 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
      301 acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa
      361 ctaatgcatg gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
      421 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
      481 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
      541 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
      601 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
      661 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
      721 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
      781 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
      841 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
      901 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
      961 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1021 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1081 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     1141 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     1201 aatctaaagt atatatgagt aacctgaggc tatggcaggg cctgccgccc cgacgttggc
     1261 tgcgagccct gggccttcac ccgaacttgg ggggtggggt ggggaaaagg aagaaacgcg
     1321 ggcgtattgg ccccaatggg gtctcggtgg ggtatcgaca gagtgccagc cctgggaccg
     1381 aaccccgcgt ttatgaacaa acgacccaac accgtgcgtt ttattctgtc tttttattgc
     1441 cgtcatagcg cgggttcctt ccggtattgt ctccttccgt gtttcagtta gcctccccct
     1501 agggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc
     1561 ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg
     1621 tgatggcagg ttgggcgtcg cttggtcggt catttcgaac cccagagtcc cgctcagaag
     1681 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa
     1741 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc
     1801 aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa
     1861 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga
     1921 tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc
     1981 tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct
     2041 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc
     2101 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac
     2161 aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca
     2221 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc
     2281 tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc
     2341 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag
     2401 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt
     2461 tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcga tctttgcaaa agcctaggcc
     2521 tccaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg cctcggcctc
     2581 tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac tgggcggagt
     2641 taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat tgagatgcat
     2701 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg ttgctgacta
     2761 attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact ttccacaccc
     2821 taactgacac acattccaca gctggttctt tccgcctcag gactcttcct ttttcaatat
     2881 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     2941 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc
     3001 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3061 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3121 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3181 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3241 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3301 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     3361 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3421 tttaacaaaa tattaacgct tacaatttac gccttaagat acattgatga gtttggacaa
     3481 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     3541 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     3601 atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa
     3661 tgtggtatgg ctgattatga tcagttatct agatccggtg gatcctcact atacccctgc
     3721 atcagtactt cgtggctgaa aaccctccct ccgaaggcta cccatgtcaa agcgggtgga
     3781 gacctcacag cgcactaata atgtgtcatc cttaatgaaa gttctttgtc ttagggcttc
     3841 cagatgcata aaagttacat agccaaaacc ttttgggttc cgtgggattg tgggtcgctg
     3901 gaaagcaagc agctctggtt tggcatccat tatctcttcg tggttttgcc ttacaggtgc
     3961 ttcagactga tcaagaattg taaggcgtat tgtaccctgg aagggccaag ggaggtggct
     4021 gtcatattct ccttgcattg tgtggacaaa aagggatata tagtttgcac agcgctgagc
     4081 agtcggtaac tgaaggtgca agcgcatgca cagtttgtac ccgggtttgc cagtgtagaa
     4141 tccagggcta tgaatcacaa caggtttctc ctcttcttga catttcaaat gcattccaaa
     4201 gttgccaatc ttccaaatat aaattccatt gcactgctgt gcttcgattt cagcaacttt
     4261 gtcctcaagg gttcgaatgg ttcgtttgag ctcacttaca tacatactct gagtttccat
     4321 tttagcagtc agctcccgga tttgatggtc ttgtcttaca aggcgaccct ctaactggtg
     4381 aatagtttcc tggaaattcc ggacctctga gatataccca gagtcgggta taacgctcaa
     4441 actatgaaca gcctgggcca acattctcat gtgtgactgg gtgttctctt gtaggtggcg
     4501 tgccaagtga ttcctctgca tcttttcatg gcaaccaaaa gtactgaatg tgcatggaat
     4561 tggggctgta gggcagtcta gatcataatg attaggcatc tgttctctga tgagtatagt
     4621 attgcagtat tcacagatga catttgccaa aggacagttc tggtcatgga tctctttatc
     4681 ttcaaatgcc attgatgcag cacagttgtc acaagaaacc tgtctccttg gacaatcctt
     4741 cagaatgtga atattaatat ggaatttttg gaagggacgc tggcattggg gacaatccat
     4801 aagagcaaac tcacaatgtg cttgatgatc ctcaagatgt ctcagttcca tcttgtgcaa
     4861 acaaccttca tttggacatt tcaccatcag agaaagaatc tcacgttttg caaaattgtc
     4921 tggaaatagt tgattttcca gcagtatttc attgtcaact ggacatttgt gacctgcatc
     4981 ccttattgat tttatgatgc aggctttgca gaacctatgg ccgcatggcg tttgcactgc
     5041 ttctcgtaat gccatcaagc agatggggca ttcatacttg ctttccaggg gtgggtcaaa
     5101 ctctacatca tatccctgga tctcctccat aaatgagctg gagaggttcc ccgtgctggc
     5161 agttccaccc acactatcat cttttgttac agcgctacag gagctggcca tggccacaca
     5221 gcagtcactt tcagactggc tggatccaca gctgttttca cagtttagca gactcatgga
     5281 gccggagccg gtaccgtcga ctgcagaatt cgaagcttgt ttttctgcac tacgcaggga
     5341 tatttcaccg cccatccagg gttttattat tccatcctgc tcaagtaata aagccccctg
     5401 tttgtctatt ccgcgtgaaa tgccaaatat ttctttatca ccaatgataa gtttcactgg
     5461 gcgattaata aaattatcca gcttttccca gcgcgacaga taaggtgcca atccttcttg
     5521 ttcgaagagt tccaacgcag cacgtaattc acgtattagc atggccgcca acgtattacg
     5581 atcgagattg atccccgctt cctgcagcgt gatccacccc tgattaacga cactctcttc
     5641 aacacggcgc attgccatgt tgatcccggc tccaatgact atttgcgccg catcgccagt
     5701 tttgccagtc agctccacca gaatgcctgc cagcttgcga tcctgcagat agaggtcatt
     5761 aggccattta acacgaactt tatctgcacc cagcttgcgt aatacttccg ccatcacgat
     5821 accgataacc agacttaaac caatcgccgc cgccgggcct tgttccagac gccagaacat
     5881 cgacaaatat aagtttgcgc caaaaggcga aaaccatttc cgaccccggc gaccacggcc
     5941 agcctgctgg tattctgcaa tgcaagcatc gcccgattta agctctccga tacgatcaag
     6001 aaggtactga ttcgtggagt caatcacagg cagcacggct acactaccgc catccagctg
     6061 acccaatatc tgtttagcat taagtaactg gataggctca ggcaggctgt atcctttacc
     6121 cggaacggta aagacatcaa cgccccagtc acgcagtgtc tgaatgtgtt tattaatagc
     6181 cgcccggctc attcccagcg tttcacccaa ctgctcgcca gagtgaaatt caccgttcgc
     6241 taacagggca atcaatttca gtggcacggt gttatccttc atccctcgag atctgagtcc
     6301 ggaagcgtag tctgggacgt cgtatgggta catggtggcg accggtagcg ctagcggatc
     6361 tgacggttca ctaaaccagc tctgcttata tagacctccc accgtacacg cctaccgccc
     6421 atttgcgtca atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc
     6481 caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac
     6541 cgctatccac gcccattgat gtactgccaa aaccgcatca cc
//
