LOCUS       sequence-273687-        4873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AID-ARF-P2A-Hygromycin fusion cassette to tag the protein of 
            interests with auxin-inducible degron and ARF at the c-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4873)
  AUTHORS   Sathyan KM, Scott TG, Guertin MJ
  TITLE     ARF-AID: A Rapidly Inducible Protein Degradation System That 
            Preserves Basal Endogenous Protein Levels.
  JOURNAL   Curr Protoc Mol Biol. 2020 Sep;132(1):e124. doi: 10.1002/cpmb.124.
  PUBMED    32757370
REFERENCE   2  (bases 1 to 4873)
  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: "Curr Protoc
            Mol Biol."; date: "2020-09"; volume: "132(1):e124. doi"; pages: " 
            10.1002/cpmb.124"
COMMENT     Sequence Label: pMGS59(AID-ARF-P2A-Hygromycin)
FEATURES             Location/Qualifiers
     source          1..4873
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(34..56)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     156..175
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     369..391
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     383..400
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     384..400
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             453..1136
                     /codon_start=1
                     /gene="Arabidopsis thaliana AXR3"
                     /product="auxin-responsive IAA17 protein"
                     /label=auxin-inducible degron
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="MGSVELNLRETELCLGLPGGDTVAPVTGNKRGFSETVDLKLNLNN
                     EPANKEGSTTHDVVTFDSKEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSG
                     GPEAAAFVKVSMDGAPYLRKIDLRMYKSYDELSNALSNMFSSFTMGKHGGEEGMIDFMN
                     ERKLMDLVNSWDYVPSYEDKDGDWMLVGDVPWPMFVDTCKRLRLMKGSDAIGLAPRAME
                     KCKSRA"
     CDS             1515..1571
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1572..2609
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     primer_bind     complement(2657..2673)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2657..2673)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2670..2692)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2681..2697
                     /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(2705..2711,2712..2729,2730..2735))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2750..2771
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2888..2905)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3059..3647)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3139..3158)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3818..4609,4610..4678))
                     /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     4441..4460
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4679..4783)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4851..4869
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
       61 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
      121 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
      181 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
      241 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
      301 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
      361 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattcgagc tcggtacctc
      421 gcgaatgcat ctagatatcg gatcccccta ggatgggcag tgtcgagctg aatctgaggg
      481 agactgagct gtgtcttggt cttcccggtg gagatacagt ggctccggta accggaaaca
      541 agagagggtt ctcagagacg gttgatctga agctaaatct gaataatgag cctgcaaaca
      601 aggaaggatc tacgactcat gacgtcgtga cttttgattc caaggagaag agtgcttgtc
      661 ctaaagatcc agccaaacct ccggccaagg cacaagttgt gggatggcca ccggtgagat
      721 cataccggaa gaacgtgatg gtttcctgcc aaaaatcaag cggtggcccg gaggcggcgg
      781 cgttcgtgaa ggtatcaatg gacggagcac cgtacttgag gaaaatcgat ttgaggatgt
      841 ataaaagcta cgatgagctt tctaatgctt tgtccaacat gttcagctct tttaccatgg
      901 gcaaacatgg aggagaagaa ggaatgatag acttcatgaa tgagaggaaa ttgatggatt
      961 tggtgaatag ctgggactat gttccctctt atgaagacaa agacggtgat tggatgctcg
     1021 tcggcgacgt tccttggcca atgttcgtcg atacatgcaa gcgtttacgt ctcatgaaag
     1081 gatcggatgc cattggtctc gctccgaggg cgatggagaa gtgcaagagc agagctagat
     1141 ctggtggatc tggtggatct ggaggttcag gtggtcccag ccaggggctg tggaacaacc
     1201 agacccagcg gatgaggaca ttcacaaagg tgcagaagcg ggggagcgtg ggccggagca
     1261 tcgatatcac caggtacagg gattacgatg agctgaggca tgatctcgcc tgtatgttcg
     1321 gcatccaggg ccagctggag gacccctacc ggatggactg gaagctggtg tacgtggacc
     1381 acgagaacga tatcctgctg gtgggcgatg acccctggga ggagttcgtg ggctgcgtga
     1441 agagcattaa gattctgtct gctgctgagg tgcagcagat gagcctggat ggagatctgg
     1501 gaggaggaag cggagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga
     1561 accctggacc tatgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     1621 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     1681 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     1741 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     1801 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     1861 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     1921 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     1981 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     2041 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     2101 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     2161 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     2221 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     2281 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     2341 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     2401 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     2461 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     2521 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     2581 cggatcggga gatgggggag gctaactgag ggcccgtcga ctgcagaggc ctgcatgcaa
     2641 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2701 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2761 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2821 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2881 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2941 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3001 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3061 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3121 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3181 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3241 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3301 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3361 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3421 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3481 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3541 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3601 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3661 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3721 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3781 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3841 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3901 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3961 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4021 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4081 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4141 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4201 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4261 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4321 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4381 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4441 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4501 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4561 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4621 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4681 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4741 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4801 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     4861 tcacgaggcc ctt
//
