LOCUS       sequence-228475-        7589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To make an auxin inducible degron mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7589)
  AUTHORS   Nishimura K, Fukagawa T
  TITLE     An efficient method to generate conditional knockout cell lines for 
            essential genes by combination of auxin-inducible degron tag and 
            CRISPR/Cas9.
  JOURNAL   Chromosome Res. 2017 Oct;25(3-4):253-260. doi: 
            10.1007/s10577-017-9559-7. Epub 2017 Jun 6.
  PUBMED    28589221
REFERENCE   2  (bases 1 to 7589)
  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: "Chromosome 
            Res."; date: "2017-10"; volume: "25(3-4):253-260. doi"; pages: " 
            10.1007/s10577-017-9559-7. Epub 2017 Jun 6"
COMMENT     Sequence Label: pAID 1.1C-T2A-Bsr
FEATURES             Location/Qualifiers
     source          1..7589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        764..1067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1068..1271
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1222..1242
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1322..3046
                     /codon_start=1
                     /product="rice transport inhibitor response 1 protein"
                     /label=OsTIR1
                     /note="F-box protein that mediates auxin-dependent 
                     interaction of a ubiquitin ligase with an auxin-inducible 
                     degron (Tan et al., 2007)"
                     /translation="MTYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIERLSRRGVFV
                     GNCYAVRAGRVAARFPNVRALTVKGKPHFADFNLVPPDWGGYAGPWIEAAARGCHGLEE
                     LRMKRMVVSDESLELLARSFPRFRALVLISCEGFSTDGLAAVASHCKLLRELDLQENEV
                     EDRGPRWLSCFPDSCTSLVSLNFACIKGEVNAGSLERLVSRSPNLRSLRLNRSVSVDTL
                     AKILLRTPNLEDLGTGNLTDDFQTESYFKLTSALEKCKMLRSLSGFWDASPVCLSFIYP
                     LCAQLTGLNLSYAPTLDASDLTKMISRCVKLQRLWVLDCISDKGLQVVASSCKDLQELR
                     VFPSDFYVAGYSAVTEEGLVAVSLGCPKLNSLLYFCHQMTNAALVTVAKNCPNFTRFRL
                     CILEPGKPDVVTSQPLDEGFGAIVRECKGLQRLSISGLLTDKVFMYIGKYAKQLEMLSI
                     AFAGDSDKGMMHVMNGCKNLRKLEIRDSPFGDAALLGNFARYETMRSLWMSSCNVTLKG
                     CQVLASKMPMLNVEVINERDGSNEMEENHGDLPKVEKLYVYRTTAGARDDAPNFVKIL"
     CDS             3047..3100
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(3527..3546)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     misc_feature    join(3610..4184,4185..4187,4188..4196)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3777..3794)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4004..4023
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4272..4958
                     /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"
     polyA_signal    5080..5201
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5117..5136)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5171..5190
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5208..5663)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5345..5366)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5557..5576
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5690..5794
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5796..6153
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5796..5987
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5819..5839)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6004..6139
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6188..6982
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             6188..6982
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6242..6261)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6852..6871
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7170..7189)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7214..7261
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
      721 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      781 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      841 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      901 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      961 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1021 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1081 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1141 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1201 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1261 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact cagatctcga
     1321 gatgacgtac ttcccggagg aggtggtgga gcacatcttc agcttcctgc cggcgcagcg
     1381 cgaccgcaac acggtctcgc tcgtctgcaa ggtgtggtac gagatcgaga ggctgagccg
     1441 ccgcggcgtc ttcgtgggca actgctacgc cgtgcgcgcc ggccgcgtcg ccgcgcggtt
     1501 ccccaacgtg cgggcgctca cggtgaaggg gaagccccac ttcgccgact tcaacctcgt
     1561 gccccccgac tggggcggct acgcggggcc gtggatcgag gcggccgcga ggggatgcca
     1621 cggcctggag gagctcagga tgaagcggat ggtggtgtcc gacgagagcc tcgagctgct
     1681 ggctcgctcg ttcccgcggt tcagggctct tgttcttatc agctgcgagg ggttcagcac
     1741 tgacgggcta gccgccgtcg cgagccattg caagcttctg agggagttgg atttgcagga
     1801 aaatgaagtg gaggatcgag ggcctaggtg gctttcctgc ttccctgatt cctgcacatc
     1861 acttgtctca ttgaattttg cctgcatcaa aggggaggtt aatgctggtt cactggagag
     1921 acttgttagc aggtccccaa acctgcggag tttgaggctg aatcgatctg tatcggtaga
     1981 tacacttgca aagatactac tgcgtacccc taacttggag gatttgggga cagggaattt
     2041 gacagatgac ttccaaactg agtcctactt taagcttacc agtgctctgg agaaatgcaa
     2101 gatgttgagg agtttgtctg gattctggga tgcttctcct gtttgcctgt catttatcta
     2161 ccccctgtgt gctcaactga caggattgaa cttgagctat gcacccacac ttgatgcttc
     2221 tgaccttaca aaaatgatta gccgctgtgt gaagctccaa cgcctttggg tactggattg
     2281 tatctcggac aaaggcttgc aagtggtggc ctccagttgc aaagacttgc aagaactcag
     2341 ggtatttcca tcagatttct acgtagctgg ttattctgca gtgacagagg agggacttgt
     2401 tgcagtatcc ttgggctgtc caaaactgaa ctcactactg tacttctgtc accaaatgac
     2461 taatgctgca ctagttactg tcgccaagaa ctgtccaaat ttcacacgat tcagactttg
     2521 tattcttgag ccagggaagc ctgatgttgt gacaagccaa ccattagatg aaggctttgg
     2581 agctattgtt cgtgagtgca agggattaca acgtttgtca atatctggtc ttctcacaga
     2641 caaagttttc atgtatattg ggaaatatgc aaaacaactt gagatgcttt ctatagcatt
     2701 tgctggtgac agtgataagg gtatgatgca tgttatgaat ggatgcaaga atttaaggaa
     2761 actggagata agagatagcc cgtttggtga tgctgcactc ttggggaatt ttgctaggta
     2821 cgagacaatg cgatcccttt ggatgtcatc ttgcaatgtc acgttaaagg ggtgccaagt
     2881 ccttgcgtca aagatgccga tgctcaatgt tgaggtcata aatgagcggg atggtagcaa
     2941 tgaaatggag gaaaaccatg gagatctgcc taaagtggag aaattatatg tgtaccgcac
     3001 aactgctggg gcgagggatg atgcaccaaa ttttgttaaa atcctagagg gccgcggcag
     3061 cctgctgacc tgcggcgacg tggaggagaa ccccggcccc atgaaaacat ttaacatttc
     3121 tcaacaagat ctagaattag tagaagtagc gacagagaag attacaatgc tttatgagga
     3181 taataaacat catgtgggag cggcaattcg tacgaaaaca ggagaaatca tttcggcagt
     3241 acatattgaa gcgtatatag gacgagtaac tgtttgtgca gaagccattg cgattggtag
     3301 tgcagtttcg aatggacaaa aggattttga cacgattgta gctgttagac acccttattc
     3361 tgacgaagta gatagaagta ttcgagtggt aagtccttgt ggtatgtgta gggagttgat
     3421 ttcagactat gcaccagatt gttttgtgtt aatagaaatg aatggcaagt tagtcaaaac
     3481 tacgattgaa gaactcattc cactcaaata tacccgaaat taaggatctt tttccctctg
     3541 ccaaaaatta tggggacatc atgaagcccc ttgaggtgga acgatcattc aagagatccc
     3601 cgggatccgc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata
     3661 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt
     3721 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct
     3781 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc
     3841 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga
     3901 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc
     3961 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt
     4021 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg
     4081 gcctcggtac acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc
     4141 gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccatgt
     4201 gcgatatcga attccgatcg acgcgtagta ctggagctgg tgcaggcgct ggagcgggtg
     4261 ccgccacaac catgggcagt gtcgagctga atctgaggga gactgagctg tgtcttggtc
     4321 ttcccggtgg agatacagtg gctccggtaa ccggaaacaa gagagggttc tcagagacgg
     4381 ttgatctgaa gctaaatctg aataatgagc ctgcaaacaa ggaaggatct acgactcatg
     4441 acgtcgtgac ttttgattcc aaggagaaga gtgcttgtcc taaagatcca gccaaacctc
     4501 cggccaaggc acaagttgtg ggatggccac cggtgagatc ataccggaag aacgtgatgg
     4561 tttcctgcca aaaatcaagc ggtggcccgg aggcggcggc gttcgtgaag gtatcaatgg
     4621 acggagcacc gtacttgagg aaaatcgatt tgaggatgta taaaagctac gatgagcttt
     4681 ctaatgcttt gtccaacatg ttcagctctt ttaccatggg caaacatgga ggagaagaag
     4741 gaatgataga cttcatgaat gagaggaaat tgatggattt ggtgaatagc tgggactatg
     4801 ttccctctta tgaagacaaa gacggtgatt ggatgctcgt cggcgacgtt ccttggccaa
     4861 tgttcgtcga tacatgcaag cgtttacgtc tcatgaaagg atcggatgcc attggtctcg
     4921 ctccgagggc gatggagaag tgcaagagca gagcttgagc ggccgcgact ctagatcata
     4981 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     5041 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     5101 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5161 cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt
     5221 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     5281 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     5341 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     5401 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     5461 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     5521 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     5581 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     5641 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5701 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5761 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
     5821 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     5881 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     5941 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     6001 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     6061 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     6121 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc
     6181 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     6241 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     6301 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     6361 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     6421 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     6481 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     6541 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     6601 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     6661 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
     6721 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     6781 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     6841 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     6901 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     6961 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     7021 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     7081 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     7141 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga
     7201 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt
     7261 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     7321 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     7381 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca
     7441 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7501 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7561 gaccccgtag aaaagatcaa aggatcttc
//
