LOCUS       sequence-204722-        7577 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 7577)
  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 7577)
  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 1.1 N-T2A-Bsr
FEATURES             Location/Qualifiers
     source          1..7577
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        54..357
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        358..561
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     512..532
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             612..2336
                     /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             2337..2390
                     /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(2817..2836)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     misc_feature    join(2900..3474,3475..3477,3478..3486)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3067..3084)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3294..3313
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3487..4170
                     /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    4358..4479
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4395..4414)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4449..4468
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4486..4941)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4623..4644)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4835..4854
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4968..5072
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5074..5431
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5074..5265
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5097..5117)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5282..5417
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5466..6260
                     /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             5466..6260
                     /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(5520..5539)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6130..6149
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6448..6467)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6492..6539
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6868..7456
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7357..7376
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
       61 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      121 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      181 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      241 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      301 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      361 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      421 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      481 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      541 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact
      601 cagatctcga gatgacgtac ttcccggagg aggtggtgga gcacatcttc agcttcctgc
      661 cggcgcagcg cgaccgcaac acggtctcgc tcgtctgcaa ggtgtggtac gagatcgaga
      721 ggctgagccg ccgcggcgtc ttcgtgggca actgctacgc cgtgcgcgcc ggccgcgtcg
      781 ccgcgcggtt ccccaacgtg cgggcgctca cggtgaaggg gaagccccac ttcgccgact
      841 tcaacctcgt gccccccgac tggggcggct acgcggggcc gtggatcgag gcggccgcga
      901 ggggatgcca cggcctggag gagctcagga tgaagcggat ggtggtgtcc gacgagagcc
      961 tcgagctgct ggctcgctcg ttcccgcggt tcagggctct tgttcttatc agctgcgagg
     1021 ggttcagcac tgacgggcta gccgccgtcg cgagccattg caagcttctg agggagttgg
     1081 atttgcagga aaatgaagtg gaggatcgag ggcctaggtg gctttcctgc ttccctgatt
     1141 cctgcacatc acttgtctca ttgaattttg cctgcatcaa aggggaggtt aatgctggtt
     1201 cactggagag acttgttagc aggtccccaa acctgcggag tttgaggctg aatcgatctg
     1261 tatcggtaga tacacttgca aagatactac tgcgtacccc taacttggag gatttgggga
     1321 cagggaattt gacagatgac ttccaaactg agtcctactt taagcttacc agtgctctgg
     1381 agaaatgcaa gatgttgagg agtttgtctg gattctggga tgcttctcct gtttgcctgt
     1441 catttatcta ccccctgtgt gctcaactga caggattgaa cttgagctat gcacccacac
     1501 ttgatgcttc tgaccttaca aaaatgatta gccgctgtgt gaagctccaa cgcctttggg
     1561 tactggattg tatctcggac aaaggcttgc aagtggtggc ctccagttgc aaagacttgc
     1621 aagaactcag ggtatttcca tcagatttct acgtagctgg ttattctgca gtgacagagg
     1681 agggacttgt tgcagtatcc ttgggctgtc caaaactgaa ctcactactg tacttctgtc
     1741 accaaatgac taatgctgca ctagttactg tcgccaagaa ctgtccaaat ttcacacgat
     1801 tcagactttg tattcttgag ccagggaagc ctgatgttgt gacaagccaa ccattagatg
     1861 aaggctttgg agctattgtt cgtgagtgca agggattaca acgtttgtca atatctggtc
     1921 ttctcacaga caaagttttc atgtatattg ggaaatatgc aaaacaactt gagatgcttt
     1981 ctatagcatt tgctggtgac agtgataagg gtatgatgca tgttatgaat ggatgcaaga
     2041 atttaaggaa actggagata agagatagcc cgtttggtga tgctgcactc ttggggaatt
     2101 ttgctaggta cgagacaatg cgatcccttt ggatgtcatc ttgcaatgtc acgttaaagg
     2161 ggtgccaagt ccttgcgtca aagatgccga tgctcaatgt tgaggtcata aatgagcggg
     2221 atggtagcaa tgaaatggag gaaaaccatg gagatctgcc taaagtggag aaattatatg
     2281 tgtaccgcac aactgctggg gcgagggatg atgcaccaaa ttttgttaaa atcctagagg
     2341 gccgcggcag cctgctgacc tgcggcgacg tggaggagaa ccccggcccc atgaaaacat
     2401 ttaacatttc tcaacaagat ctagaattag tagaagtagc gacagagaag attacaatgc
     2461 tttatgagga taataaacat catgtgggag cggcaattcg tacgaaaaca ggagaaatca
     2521 tttcggcagt acatattgaa gcgtatatag gacgagtaac tgtttgtgca gaagccattg
     2581 cgattggtag tgcagtttcg aatggacaaa aggattttga cacgattgta gctgttagac
     2641 acccttattc tgacgaagta gatagaagta ttcgagtggt aagtccttgt ggtatgtgta
     2701 gggagttgat ttcagactat gcaccagatt gttttgtgtt aatagaaatg aatggcaagt
     2761 tagtcaaaac tacgattgaa gaactcattc cactcaaata tacccgaaat taaggatctt
     2821 tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgaggtgga acgatcattc
     2881 aagagatccc cgggatccgc ccctctccct cccccccccc taacgttact ggccgaagcc
     2941 gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt
     3001 ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc
     3061 tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc
     3121 tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc
     3181 cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg
     3241 cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct
     3301 cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat
     3361 ctgatctggg gcctcggtac acatgcttta catgtgttta gtcgaggtta aaaaaacgtc
     3421 taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc
     3481 acaaccatgg gcagtgtcga gctgaatctg agggagactg agctgtgtct tggtcttccc
     3541 ggtggagata cagtggctcc ggtaaccgga aacaagagag ggttctcaga gacggttgat
     3601 ctgaagctaa atctgaataa tgagcctgca aacaaggaag gatctacgac tcatgacgtc
     3661 gtgacttttg attccaagga gaagagtgct tgtcctaaag atccagccaa acctccggcc
     3721 aaggcacaag ttgtgggatg gccaccggtg agatcatacc ggaagaacgt gatggtttcc
     3781 tgccaaaaat caagcggtgg cccggaggcg gcggcgttcg tgaaggtatc aatggacgga
     3841 gcaccgtact tgaggaaaat cgatttgagg atgtataaaa gctacgatga gctttctaat
     3901 gctttgtcca acatgttcag ctcttttacc atgggcaaac atggaggaga agaaggaatg
     3961 atagacttca tgaatgagag gaaattgatg gatttggtga atagctggga ctatgttccc
     4021 tcttatgaag acaaagacgg tgattggatg ctcgtcggcg acgttccttg gccaatgttc
     4081 gtcgatacat gcaagcgttt acgtctcatg aaaggatcgg atgccattgg tctcgctccg
     4141 agggcgatgg agaagtgcaa gagcagagct gcggccggag ctggtgcagg cgctggagcg
     4201 ggtgccgata tcgaattccg atcgacgcgt agtactgcgg ccgcgactct agatcataat
     4261 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     4321 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     4381 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4441 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
     4501 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     4561 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     4621 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     4681 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     4741 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     4801 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     4861 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     4921 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4981 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5041 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     5101 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     5161 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     5221 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     5281 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     5341 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     5401 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     5461 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     5521 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     5581 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     5641 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     5701 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     5761 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     5821 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     5881 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     5941 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     6001 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     6061 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     6121 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     6181 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     6241 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     6301 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     6361 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     6421 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     6481 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     6541 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     6601 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     6661 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     6721 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6781 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6841 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6901 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6961 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7021 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7081 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7141 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7201 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7261 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7321 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7381 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7441 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7501 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     7561 cgccatgcat tagttat
//
