LOCUS       sequence-366751-        6722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses V5-tagged GlycoID construct: GafD_short-miniTurboID in 
            mammalian cytosol for O-GlcNAc interactome tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6722)
  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 6722)
  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: cyt-GafD-mTurboID-V5
FEATURES             Location/Qualifiers
     source          1..6722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        60..439
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        440..643
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     594..614
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     688..707
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        688..706
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1203..1212
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1212..1253
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1260..2030
                     /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             2037..2063
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(2100..2118)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2101..2118)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2138..2155)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2144..2368
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2414..2842
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2501..2520)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2711..2732
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2851..2871)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2856..3185
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3036..3171
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3098..3117
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3252..4046
                     /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(3306..3325)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3916..3935
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4220..4341
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4257..4276)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4311..4330
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4390..4406)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4390..4406)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4403..4425)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4414..4430
                     /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(4438..4444,4445..4462,4463..4468))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4483..4504
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4621..4638)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4792..5380)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4872..4891)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5551..6342,6343..6411))
                     /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(6412..6516)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6591..6610)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
       61 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      121 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      181 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      421 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      481 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      541 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      601 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
      661 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctta
      721 tggccgtgtc cttcatcggc agcaccgaaa atgatgtggg ccctagccag ggcagctaca
      781 gctctacaca cgccatggac aacctgcctt tcgtgtacaa caccggctac aatatcggct
      841 accagaacgc caacgtgtgg cggatctctg gcggcttttg tgttggcctg gacggcaaag
      901 tggatctgcc tgttgtgggc tctctggacg gccagtctat ctacggcctg acagaggaag
      961 tgggcctgct gatctggatg ggcgacacca attacagcag aggcacagcc atgagcggca
     1021 acagctggga gaatgtgttc agcggatggt gcgtgggcaa ctacgtgtcc acacagggac
     1081 tgtctgtgca cgtgcggcct gtgatcctga agagaaatag cagcgcccag tacagcgtgc
     1141 agaaaaccag catcggctcc atcagaatgc ggccctacaa tggcagctct aagcttgcgg
     1201 ccgccaccat gggcaagccc atccccaacc ccctgctggg cctggacagc accgctagca
     1261 tcccgctgct gaacgctaaa cagattctgg gacagctgga cggcgggagc gtggcagtcc
     1321 tgcctgtggt cgactccacc aatcagtacc tgctggatcg aatcggcgag ctgaagagtg
     1381 gggatgcttg cattgcagaa tatcagcagg cagggagagg aagcagaggg aggaaatggt
     1441 tctctccttt tggagctaac ctgtacctga gtatgttttg gcgcctgaag cggggaccag
     1501 cagcaatcgg cctgggcccg gtcatcggaa ttgtcatggc agaagcgctg cgaaagctgg
     1561 gagcagacaa ggtgcgagtc aaatggccca atgacctgta tctgcaggat agaaagctgg
     1621 caggcatcct ggtggagctg gccggaataa caggcgatgc tgcacagatc gtcattggcg
     1681 ccgggattaa cgtggctatg aggcgcgtgg aggaaagcgt ggtcaatcag ggctggatca
     1741 cactgcagga agcagggatt aacctggaca ggaatactct ggccgctatg ctgatccgag
     1801 agctgcgggc agccctggaa ctgttcgagc aggaaggcct ggctccatat ctgtcacggt
     1861 gggagaagct ggataacttc atcaatagac ccgtgaagct gatcattggg gacaaagaga
     1921 ttttcgggat tagccggggg attgataaac agggagccct gctgctggaa caggacggag
     1981 ttatcaaacc ctggatgggc ggagaaatca gtctgcggtc tgccgaaaag ctgcagctgc
     2041 ctcccctgga gcgcctgacc ctggactaat agctcgagca tgcatctaga gggccctatt
     2101 ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     2161 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2221 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2281 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2341 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     2401 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2461 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2521 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2581 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2641 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2701 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2761 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2821 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     2881 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2941 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3001 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3061 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3121 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3181 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     3241 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3301 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3361 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3421 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3481 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3541 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     3601 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     3661 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     3721 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     3781 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     3841 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     3901 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     3961 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4021 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4081 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4141 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4201 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     4261 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4321 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     4381 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4441 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4501 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4561 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4621 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4681 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4741 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4801 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4861 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4921 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4981 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5041 ctgtgtgcac aaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5101 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5161 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5221 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5281 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5341 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5401 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5461 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5521 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5581 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5641 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5701 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5761 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5821 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5881 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5941 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6001 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6061 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6121 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6181 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6241 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6301 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6361 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6421 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6481 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6541 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     6601 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     6661 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     6721 aa
//
