LOCUS       sequence-422262-        4956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For ORF tagging with IAA17-4Flag. IAA17 is an auxin-inducible degron
            (AID). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4956)
  AUTHORS   He CW, Cui XF, Ma SJ, Xu Q, Ran YP, Chen WZ, Mu JX, Li H, Zhu J, 
            Gong Q, Xie Z
  TITLE     Membrane recruitment of Atg8 by Hfl1 facilitates turnover of 
            vacuolar membrane proteins in yeast cells approaching stationary 
            phase.
  JOURNAL   BMC Biol. 2021 Jun 4;19(1):117. doi: 10.1186/s12915-021-01048-7.
  PUBMED    34088313
REFERENCE   2  (bases 1 to 4956)
  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: "BMC Biol.";
            date: "2021-06-4"; volume: "19(1):117. doi"; pages: " 
            10.1186/s12915-021-01048-7"
COMMENT     Sequence Label: IAA17-4flag-HIS5
FEATURES             Location/Qualifiers
     source          1..4956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             63..749
                     /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="MMGSVELNLRETELCLGLPGGDTVAPVTGNKRGFSETVDLKLNLN
                     NEPANKEGSTTHDVVTFDSKEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSS
                     GGPEAAAFVKVSMDGAPYLRKIDLRMYKSYDELSNALSNMFSSFTMGKHGGEEGMIDFM
                     NERKLMDLVNSWDYVPSYEDKDGDWMLVGDVPWPMFVDTCKRLRLMKGSDAIGLAPRAM
                     EKCKSRA"
     CDS             777..800
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             807..830
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             837..860
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             867..890
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      920..1107
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(975..999)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    1159..1192
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            1247..2447
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5 
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        1247..1590
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1591..2244
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe his5"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=S. pombe his5
                     /note="yeast auxotrophic marker; corresponds to S. 
                     cerevisiae HIS3"
                     /translation="MGRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHANQ
                     KGEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQ
                     AMGNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLY
                     SFSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     terminator      2250..2447
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    2507..2540
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2593..2612)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2594..2612)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2699..2716)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2870..3458)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2950..2969)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3629..4420,4421..4489))
                     /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     4252..4271
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4490..4594)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4662..4680
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4718..4740)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4840..4859
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4940..4956,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4940..4956,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg tgggattcct gggttaatta
       61 acatgatggg cagtgtcgag ctgaatctga gggagactga gctgtgtctt ggtcttcccg
      121 gtggagatac agtggctccg gtaaccggaa acaagagagg gttctcagag acggttgatc
      181 tgaagctaaa tctgaataat gagcctgcaa acaaggaagg atctacgact catgacgtcg
      241 tgacttttga ttccaaggag aagagtgctt gtcctaaaga tccagccaaa cctccggcca
      301 aggcacaagt tgtgggatgg ccaccggtga gatcataccg gaagaacgtg atggtttcct
      361 gccaaaaatc aagcggtggc ccggaggcgg cggcgttcgt gaaggtatca atggacggag
      421 caccgtactt gaggaaaatc gatttgagga tgtataaaag ctacgatgag ctttctaatg
      481 ctttgtccaa catgttcagc tcttttacca tgggcaaaca tggaggagaa gaaggaatga
      541 tagacttcat gaatgagagg aaattgatgg atttggtgaa tagctgggac tatgttccct
      601 cttatgaaga caaagacggt gattggatgc tcgtcggcga cgttccttgg ccaatgttcg
      661 tcgatacatg caagcgttta cgtctcatga aaggatcgga tgccattggt ctcgctccga
      721 gggcgatgga gaagtgcaag agcagagctg ctagcgggtt aattaacatc gctagcgatt
      781 ataaagatga tgatgataaa cctagcgatt ataaagatga tgatgataaa cctagcgatt
      841 ataaagatga tgatgataaa cctagcgatt ataaagatga tgatgataaa cctaggtgag
      901 gcgcgccact tctaaataag cgaatttctt atgatttatg atttttatta ttaaataagt
      961 tataaaaaaa ataagtgtat acaaatttta aagtgactct taggttttaa aacgaaaatt
     1021 cttattcttg agtaactctt tcctgtaggt caggttgctt tctcaggtat agtatgaggt
     1081 cgctcttatt gaccacacct ctaccggcag atccgctagg gataacaggg taatatctgc
     1141 aggtcgacaa cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtcta
     1201 gagatctgtt tagcttgcct cgtccccgcc gggtcacccg gccagcgaca tggaggccca
     1261 gaataccctc cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc
     1321 gtacatttag cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg
     1381 cacggcgcga agcaaaaatt acggctcctc gctgcagacc tgcgagcagg gaaacgctcc
     1441 cctcacagac gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta
     1501 ggatttgcca ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag
     1561 ttctcacatc acatccgaac ataaacaacc atgggtagga gggcttttgt agaaagaaat
     1621 acgaacgaaa cgaaaatcag cgttgccatc gctttggaca aagctccctt acctgaagag
     1681 tcgaatttta ttgatgaact tataacttcc aagcatgcaa accaaaaggg agaacaagta
     1741 atccaagtag acacgggaat tggattcctg gatcacatgt atcatgcact ggctaaacat
     1801 gcaggctgga gcttacgact ttactcaaga ggtgatttaa tcatcgatga tcatcacact
     1861 gcagaagata ctgctattgc acttggtatt gcattcaagc aggctatggg taactttgcc
     1921 ggcgttaaaa gatttggaca tgcttattgt ccacttgacg aagccctttc tagaagcgta
     1981 gttgacttgt cgggacggcc ctatgctgtt atcgatttgg gattaaagcg tgaaaaggtt
     2041 ggggaattgt cctgtgaaat gatccctcac ttactatatt ccttttcggt agcagctgga
     2101 attactttgc atgttacctg cttatatggt agtaatgacc atcatcgtgc tgaaagcgct
     2161 tttaaatctc tggctgttgc catgcgcgcg gctactagtc ttactggaag ttctgaagtc
     2221 ccaagcacga agggagtgtt gtaaagagta ctgacaataa aaagattctt gttttcaaga
     2281 acttgtcatt tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg
     2341 tgatttatat tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga
     2401 aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg tcgattcgat
     2461 actaacgccg ccatccagtt taaacgagct ctcgagaacc cttaatataa cttcgtataa
     2521 tgtatgctat acgaagttat taggtgatat cagatccact agtggcctat gcggccgcgg
     2581 atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt aacctgcatt
     2641 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2701 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2761 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2821 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2881 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2941 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3001 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3061 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3121 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3181 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3241 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3301 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3361 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3421 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3481 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3541 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3601 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3661 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3721 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3781 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3841 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3901 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3961 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4021 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4081 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4141 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4201 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4261 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4321 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4381 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4441 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4501 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4561 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4621 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4681 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     4741 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     4801 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     4861 tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa tacataacct
     4921 tatgtatcat acacatacga tttaggtgac actata
//
