LOCUS       sequence-464733-        7304 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express BirA tagged to the C-terminus of CHIP with a GSGS linker 
            between BirA and CHIP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7304)
  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 7304)
  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-CHIP-GSGS-BirA-HA
FEATURES             Location/Qualifiers
     source          1..7304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1680..1699)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1681..1699)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1744..1947)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1773..1793)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1948..2327
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2499..2518
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2593..2697
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2698..2766,2767..3558)
                     /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(2916..2935)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3729..4317
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4218..4237
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4471..4488
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4605..4626
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4641..4646,4647..4664,4665..4671)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4679..4695
                     /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     4684..4706
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4703..4719
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4703..4719
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4768..4889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4779..4798)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4833..4852
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5063..5857)
                     /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
                     GLAPAELFARLKARMPNGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5174..5193)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5784..5803
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5924..6253)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5938..6073
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5992..6011)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     6238..6258
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(6267..6695)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6377..6398)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6589..6608
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6741..6965
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6954..6971
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(7002..7028)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(join(7032..7304,1..687))
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
ORIGIN
        1 gccagcctcc tgcagggtga tccagccctg gttcaccacg ctctcctcca ctcttctcat
       61 ggccatgttg attccggctc cgatcacgat ctgagcggca tcgcctgtct tgcctgtcag
      121 ctccaccagg atgccagcca gcttgcggtc ctggaggtac aggtcattag gccacttcac
      181 tctcaccttg tcggctccca gctttctcag cacctcggcc atcacgattc cgatcaccag
      241 actcagtccg atggcagcag caggtccctg ctccagtctc cagaacatgg acaggtacag
      301 gttggctccg aatgggctga accacttccg tcctctgcgt cctctgccag cctgctggta
      361 ctcggcgatg caggcgtcgc cggacttcag ctctccgatt ctgtccagca ggtactgatt
      421 ggtggagtcg atcacaggca gcacggccac gcttccgcca tccagctgtc ccaggatctg
      481 cttggcgttc agcagctgga taggctcagg caggctgtag cccttgccag gcacggtgaa
      541 cacgtccact ccccagtctc tcagtgtctg gatgtgcttg ttgatggcgg ctctgctcat
      601 tcccagggtc tctcccagct gctcgccaga gtggaactcg ccgttggcca gcagggcgat
      661 cagcttcagg ggcacggtgt tgtccttaga gccagagccg gatccgaatt cgaatccgga
      721 gactaagtag tcctccaccc agccattctc agagatgaat gcgtcaataa cctccttcat
      781 agccaagttg gggatgagct gttcctgggt cagggggctc cgggtcacgg ggtcaaaatg
      841 acccacacgc tgcaggtgct cctcgatgtc cttgcggtcg taggtgatgc cactgggcgt
      901 gatgcacggc tcccgcatca gctcaaagct gatcttgcca cacaggtagt cggggatgtc
      961 tcgcttcttc ctcttctcat ccacctgaga aaaaagctcg tccatgtccg ccatgtactt
     1021 gtcgtgcttg gcctcaatgc aggcctgctg ggcccggacg tggctgtcgt cctcatcacc
     1081 ctcgtggttt cgctggcact cttccagctc cctctcacgc tccgcggcaa tgagcctgga
     1141 gaggtaggag tgcagctcgc tctcctggtg gatgcgccgc tcctcaatgc tgttccagcg
     1201 cttcttcttc gcgattcgaa gagcgctggg gatgtcgtcc ccgaagttca gccgctgctc
     1261 cttggccagg ctgtaagctc gctgcagatt ggcgatggcc tcatcatagc tctccatctc
     1321 cagctggcac tgccccagga agaagtgcgc cttcacagac tgcccgtcca gctccagggc
     1381 gcgccggcag tcggccaggg cctgctcgtg ctgctgcatc ttcaggtagc acaaggcccg
     1441 gttggtgtaa tacacggcca ccagcgggtt ccgggtgatc gcgcggccgt agcaggccgc
     1501 cgcctccggg tactttcggc ccacgaacag acgattgccc tgctccttga gctcctgcgc
     1561 gctcgggctc ttctcggggc ttccgccgcc agcgcccagc cgtgcgccgc cctccttctc
     1621 ctccttgccc ttcatgtacg accggttaac aggccttaag cgctagccag cttgggtctc
     1681 cctatagtga gtcgtattaa tttcgataag ccagtaagca gtgggttctc tagttagcca
     1741 gagagctctg cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg
     1801 ggcggagttg ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc
     1861 attgacgtca atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc
     1921 attgatgtac tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta
     1981 ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt
     2041 accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa
     2101 gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt
     2161 tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc
     2221 aggcgggcca tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa
     2281 tgaccccgta attgattact attaataact agtcaataat caatgtcaac gcgtatatct
     2341 ggcccgtaca tcgcgaagca gcgcaaaacg cctaacccta agcagattct tcatgcaatt
     2401 gtcggtcaag ccttgccttg ttgtagctta aattttgctc gcgcactact cagcgacctc
     2461 caacacacaa gcagggagca gatactggct taactatgcg gcatcagagc agattgtact
     2521 gagagtgcac cataggggat cgggagatct cccgatccgt cgacgtcagg tggcactttt
     2581 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2641 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2701 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     2761 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     2821 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     2881 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     2941 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3001 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3061 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3121 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3181 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3241 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3301 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3361 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3421 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3481 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3541 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3601 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3661 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3721 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3781 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3841 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     3901 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3961 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4021 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4081 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4141 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4201 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4261 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4321 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4381 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4441 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4501 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     4561 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     4621 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     4681 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagctctag
     4741 ctagaggtcg acggtataca gacatgataa gatacattga tgagtttgga caaaccacaa
     4801 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     4861 taaccattat aagctgcaat aaacaagttg gggtgggcga agaactccag catgagatcc
     4921 ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca
     4981 tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc
     5041 atttcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc
     5101 gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc
     5161 caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac
     5221 ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca
     5281 agcaggcatc gccatgggtc acgacgagat cctcgccgtt gggcatgcgc gccttgagcc
     5341 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
     5401 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
     5461 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata
     5521 ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata
     5581 gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg
     5641 tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca
     5701 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat
     5761 cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg
     5821 ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct
     5881 cttgatcaga tccgaaaatg gatatacaag ctcccgggag ctttttgcaa aagcctaggc
     5941 ctccaaaaaa gcctcctcac tacttctgga atagctcaga ggcagaggcg gcctcggcct
     6001 ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa ctgggcggag
     6061 ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca
     6121 tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact
     6181 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
     6241 ctaactgaca cacattccac agaattaatt cgcgttaaat ttttgttaaa tcagctcatt
     6301 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     6361 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     6421 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     6481 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     6541 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     6601 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     6661 acccgccgcg cttaatgcgc cgctacaggg cgcgtgggga taccccctag agccccagct
     6721 ggttctttcc gcctcagaag ccatagagcc caccgcatcc ccagcatgcc tgctattgtc
     6781 ttcccaatcc tcccccttgc tgtcctgccc caccccaccc cccagaatag aatgacacct
     6841 actcagacaa tgcgatgcaa tttcctcatt ttattaggaa aggacagtgg gagtggcacc
     6901 ttccagggtc aaggaaggca cgggggaggg gcaaacaaca gatggctggc aactagaagg
     6961 cacagtcgag gctgatcagc ggtttaaact taggccgcct atgcgtaatc cggtacatcg
     7021 taagggtaag ccttctctgc gcttctcagg gagatttctc cgcccatcca gggcttgatg
     7081 attccgtcct gctccagcag cagagctccc tgcttgtcga ttcctctgga gatgccgaag
     7141 atttccttgt cgccgatgat cagcttcaca ggtctgttga tgaagttgtc cagcttctcc
     7201 catctgctca ggtaaggagc cagtccctcc tgctcgaaca gctccagagc ggctctcagc
     7261 tctctgatca gcatggcggc cagggtgttc cggtccaggt tgat
//
