LOCUS       sequence-266718-        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(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             448..1131
                     /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             1510..1566
                     /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             1567..2604
                     /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(2652..2668)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2652..2668)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2665..2687)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2676..2692
                     /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(2700..2706,2707..2724,2725..2730))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2745..2766
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2883..2900)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3054..3642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3134..3153)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3813..4604,4605..4673))
                     /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     4436..4455
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4674..4778)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4846..4864
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tatcggatcc ccctaggatg ggcagtgtcg agctgaatct gagggagact
      481 gagctgtgtc ttggtcttcc cggtggagat acagtggctc cggtaaccgg aaacaagaga
      541 gggttctcag agacggttga tctgaagcta aatctgaata atgagcctgc aaacaaggaa
      601 ggatctacga ctcatgacgt cgtgactttt gattccaagg agaagagtgc ttgtcctaaa
      661 gatccagcca aacctccggc caaggcacaa gttgtgggat ggccaccggt gagatcatac
      721 cggaagaacg tgatggtttc ctgccaaaaa tcaagcggtg gcccggaggc ggcggcgttc
      781 gtgaaggtat caatggacgg agcaccgtac ttgaggaaaa tcgatttgag gatgtataaa
      841 agctacgatg agctttctaa tgctttgtcc aacatgttca gctcttttac catgggcaaa
      901 catggaggag aagaaggaat gatagacttc atgaatgaga ggaaattgat ggatttggtg
      961 aatagctggg actatgttcc ctcttatgaa gacaaagacg gtgattggat gctcgtcggc
     1021 gacgttcctt ggccaatgtt cgtcgataca tgcaagcgtt tacgtctcat gaaaggatcg
     1081 gatgccattg gtctcgctcc gagggcgatg gagaagtgca agagcagagc tagatctggt
     1141 ggatctggtg gatctggagg ttcaggtggt cccagccagg ggctgtggaa caaccagacc
     1201 cagcggatga ggacattcac aaaggtgcag aagcggggga gcgtgggccg gagcatcgat
     1261 atcaccaggt acagggatta cgatgagctg aggcatgatc tcgcctgtat gttcggcatc
     1321 cagggccagc tggaggaccc ctaccggatg gactggaagc tggtgtacgt ggaccacgag
     1381 aacgatatcc tgctggtggg cgatgacccc tgggaggagt tcgtgggctg cgtgaagagc
     1441 attaagattc tgtctgctgc tgaggtgcag cagatgagcc tggatggaga tctgggagga
     1501 ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct
     1561 ggacctatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
     1621 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
     1681 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
     1741 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
     1801 gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc
     1861 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc
     1921 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
     1981 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
     2041 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
     2101 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
     2161 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
     2221 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
     2281 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
     2341 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
     2401 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
     2461 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
     2521 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccggat
     2581 cgggagatgg gggaggctaa ctgagggccc gtcgactgca gaggcctgca tgcaagcttg
     2641 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2701 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2761 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2821 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2881 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2941 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3001 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3061 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3121 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3181 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3241 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3301 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3361 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3421 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3481 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3541 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3601 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3661 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3721 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3781 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3841 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3901 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3961 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4021 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4081 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4141 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4201 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4261 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4321 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4381 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4441 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4501 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4561 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4621 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4681 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4741 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4801 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     4861 aggccctttc gtc
//
