LOCUS       sequence-366747-        6809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xHA-tagged GlycoID construct: GafD_short-miniTurboID in 
            mammalian nucleus for O-GlcNAc interactome tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6809)
  AUTHORS   Liu Y, Nelson ZM, Reda A, Fehl C
  TITLE     Spatiotemporal Proximity Labeling Tools to Track GlcNAc 
            Sugar-Modified Functional Protein Hubs during Cellular Signaling.
  JOURNAL   ACS Chem Biol. 2022 Jul 12. doi: 10.1021/acschembio.2c00282.
  PUBMED    35819414
REFERENCE   2  (bases 1 to 6809)
  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: "ACS Chem 
            Biol. 2022 Jul 12. doi: 10.1021/acschembio.2c00282."
COMMENT     Sequence Label: nuc-GafD-mTurboID-HA
FEATURES             Location/Qualifiers
     source          1..6809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        129..508
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        509..712
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     663..683
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     757..776
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        757..775
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1275..1301
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1305..1331
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1335..1361
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1368..2138
                     /codon_start=1
                     /product="N-terminally truncated mutant of the E. coli 
                     biotin ligase BioID engineered for improved catalytic 
                     efficiency (Branon et al., 2018)"
                     /label=miniTurbo
                     /translation="IPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACI
                     AEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADK
                     VRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQ
                     EAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIF
                     GISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             2157..2177
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2181..2201
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2205..2225
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(2256..2274)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2257..2274)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2294..2311)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2300..2524
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2570..2998
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2657..2676)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2867..2888
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3007..3027)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3012..3341
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3192..3327
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3254..3273
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3408..4202
                     /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(3462..3481)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4072..4091
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4376..4497
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4413..4432)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4467..4486
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4546..4562)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4546..4562)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4559..4581)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4570..4586
                     /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)."
     promoter        complement(join(4594..4600,4601..4618,4619..4624))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4639..4660
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4777..4794)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4948..5536)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5028..5047)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5707..6498,6499..6567))
                     /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"
     promoter        complement(6568..6672)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6747..6766)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
       61 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
      121 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      181 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      241 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      301 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      361 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      421 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      481 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      541 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      601 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      661 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct
      721 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac
      781 ccaagcttat ggccgtgtcc ttcatcggca gcaccgaaaa tgatgtgggc cctagccagg
      841 gcagctacag ctctacacac gccatggaca acctgccttt cgtgtacaac accggctaca
      901 atatcggcta ccagaacgcc aacgtgtggc ggatctctgg cggcttttgt gttggcctgg
      961 acggcaaagt ggatctgcct gttgtgggct ctctggacgg ccagtctatc tacggcctga
     1021 cagaggaagt gggcctgctg atctggatgg gcgacaccaa ttacagcaga ggcacagcca
     1081 tgagcggcaa cagctgggag aatgtgttca gcggatggtg cgtgggcaac tacgtgtcca
     1141 cacagggact gtctgtgcac gtgcggcctg tgatcctgaa gagaaatagc agcgcccagt
     1201 acagcgtgca gaaaaccagc atcggctcca tcagaatgcg gccctacaat ggcagctctg
     1261 cggccgccac catgtacccg tatgatgttc cggattacgc tggctatccc tacgacgtgc
     1321 ccgactatgc cgggtacccc tatgacgtcc cagactacgc agctagcatc ccgctgctga
     1381 acgctaaaca gattctggga cagctggacg gcgggagcgt ggcagtcctg cctgtggtcg
     1441 actccaccaa tcagtacctg ctggatcgaa tcggcgagct gaagagtggg gatgcttgca
     1501 ttgcagaata tcagcaggca gggagaggaa gcagagggag gaaatggttc tctccttttg
     1561 gagctaacct gtacctgagt atgttttggc gcctgaagcg gggaccagca gcaatcggcc
     1621 tgggcccggt catcggaatt gtcatggcag aagcgctgcg aaagctggga gcagacaagg
     1681 tgcgagtcaa atggcccaat gacctgtatc tgcaggatag aaagctggca ggcatcctgg
     1741 tggagctggc cggaataaca ggcgatgctg cacagatcgt cattggcgcc gggattaacg
     1801 tggctatgag gcgcgtggag gaaagcgtgg tcaatcaggg ctggatcaca ctgcaggaag
     1861 cagggattaa cctggacagg aatactctgg ccgctatgct gatccgagag ctgcgggcag
     1921 ccctggaact gttcgagcag gaaggcctgg ctccatatct gtcacggtgg gagaagctgg
     1981 ataacttcat caatagaccc gtgaagctga tcattgggga caaagagatt ttcgggatta
     2041 gccgggggat tgataaacag ggagccctgc tgctggaaca ggacggagtt atcaaaccct
     2101 ggatgggcgg agaaatcagt ctgcggtctg ccgaaaagga attcagcagg gccgacccca
     2161 agaagaagag gaaggtggac cccaagaaga agaggaaggt ggaccccaag aagaagagga
     2221 aggtgtgact cgagcatgca tctagagggc cctattctat agtgtcacct aaatgctaga
     2281 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2341 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2401 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2461 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     2521 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     2581 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2641 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2701 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2761 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2821 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2881 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2941 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     3001 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3061 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3121 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3181 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3241 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     3301 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     3361 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3421 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3481 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3541 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     3601 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3661 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3721 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3781 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3841 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3901 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     3961 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4021 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4081 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4141 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4201 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4261 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4321 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4381 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4441 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4501 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     4561 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4621 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4681 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4741 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4801 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4861 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4921 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4981 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5041 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5101 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5161 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5221 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5281 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5341 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5401 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5461 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5521 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5581 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5641 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5701 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5761 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5821 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5881 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5941 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6001 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6061 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6121 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6181 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6241 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6301 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6361 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6421 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6481 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6541 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6601 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6661 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
     6721 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     6781 tatctgctcc ctgcttgtgt gttggaggt
//
