LOCUS       sequence-291421-        7593 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of OsTIR1 and GFP-mAID protein in mammalian cells under 
            the control of EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7593)
  AUTHORS   Nishimura K, Yamada R, Hagihara S, Iwasaki R, Uchida N, Kamura T, 
            Takahashi K, Torii KU, Fukagawa T
  TITLE     A super-sensitive auxin-inducible degron system with an engineered 
            auxin-TIR1 pair.
  JOURNAL   Nucleic Acids Res. 2020 Oct 9;48(18):e108. doi: 10.1093/nar/gkaa748.
  PUBMED    32941625
REFERENCE   2  (bases 1 to 7593)
  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: "Nucleic 
            Acids Res."; date: "2020-10-9"; volume: "48(18):e108. doi"; pages: "
            10.1093/nar/gkaa748"
COMMENT     Sequence Label: pAID1.2-EF1a-NGFP-mAID
FEATURES             Location/Qualifiers
     source          1..7593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             51..1775
                     /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             1776..1829
                     /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(2256..2275)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     misc_feature    join(2312..2886,2887..2889,2890..2898)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2479..2496)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2706..2725
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2911..2913,2914..2916,2917..3627)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2911..2913,2914..2916,2917..3627)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2956..2977)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3217..3236)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3564..3585
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3628..3831
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     polyA_signal    3882..4003
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3919..3938)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3973..3992
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4157..4745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4237..4256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4972..5763,5764..5832))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     polyA_signal    5937..5985
                     /note="synthetic polyadenylation signal"
     misc_feature    5999..6090
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6039..6058
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        6110..6320
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        6132..6328
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6179..6314
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        6415..7593
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6646..7584
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7541..7561
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 attgcggaag gccgtcaagg ccacgtgtct tgtccagagg cgctgccacc atgacatact
       61 ttcctgaaga ggtcgtcgaa cacattttta gcttcctgcc tgcacagaga gatagaaaca
      121 cagtgagcct ggtctgcaaa gtgtggtacg agatcgaacg cctgagccgg agaggagtgt
      181 tcgtcggcaa ctgctatgct gtgagagcag gcagggtcgc cgctaggttt ccaaatgtgc
      241 gcgcactgac cgtcaagggg aaaccccact tcgccgactt taacctggtg ccccctgatt
      301 ggggaggata cgccggccct tggatcgagg cagccgctcg cggctgtcat ggactggagg
      361 aactgcgcat gaagcgaatg gtggtctctg acgaaagtct ggagctgctg gctcggagct
      421 tccctaggtt tcgcgcactg gtgctgattt cttgcgaagg cttcagcacc gatggactgg
      481 cagccgtggc ctcccactgt aagctgctgc gggagctgga cctccaggag aatgaagtgg
      541 aggatagagg ccccagatgg ctgtcttgct tcccagactc atgtaccagc ctggtgtccc
      601 tgaactttgc ctgcatcaaa ggcgaagtga atgctgggtc cctggagcgg ctggtctcaa
      661 gaagccccaa cctgaggtct ctgcggctga accggagcgt gagcgtggac actctggcta
      721 agattctgct gagaacccct aacctggagg atctgggaac cggcaatctg acagacgatt
      781 tccagacaga atcctacttt aaactgactt ctgccctgga gaagtgtaaa atgctgagga
      841 gtctgtcagg attctgggat gcttcacccg tgtgcctgag ctttatctac cctctgtgtg
      901 cacagctgac aggcctgaac ctgagctatg caccaaccct ggacgccagt gatctgacaa
      961 agatgatctc acgctgcgtg aaactccagc gactgtgggt gctggactgt atttccgata
     1021 aggggctcca ggtggtcgcc agctcctgca aggacctcca ggagctgaga gtgttcccat
     1081 ctgattttta cgtggccgga tatagtgctg tcactgagga aggcctggtg gcagtctcac
     1141 tgggatgccc aaagctgaac agcctgctgt atttctgtca tcagatgact aatgctgcac
     1201 tggtgaccgt cgccaagaac tgccctaatt tcacccgatt tcggctgtgt attctggaac
     1261 caggcaaacc cgacgtggtc acatcccagc cactggatga agggtttgga gctatcgtga
     1321 gagagtgcaa gggactccag aggctgagca tttccggcct gctgacagac aaagtgttca
     1381 tgtacatcgg caagtatgct aagcagctgg agatgctgag cattgcattt gccggagact
     1441 ccgataaggg catgatgcac gtgatgaacg ggtgtaagaa tctgcgaaaa ctggaaatcc
     1501 gggacagccc tttcggggat gccgctctgc tgggaaactt tgccagatac gagacaatga
     1561 ggagcctgtg gatgtctagt tgcaatgtga ctctgaaggg ctgtcaggtc ctggctagta
     1621 aaatgcctat gctgaacgtg gaagtcatta atgagcggga cgggtctaac gaaatggagg
     1681 aaaatcatgg cgacctgcca aaggtggaga aactgtatgt gtatcggacc accgcagggg
     1741 caagagatga tgctcccaac tttgtgaaga ttctggaggg ccgcggcagc ctgctgacct
     1801 gcggcgacgt ggaggagaac cccggcccca tgaaaacatt taacatttct caacaagatc
     1861 tagaattagt agaagtagcg acagagaaga ttacaatgct ttatgaggat aataaacatc
     1921 atgtgggagc ggcaattcgt acgaaaacag gagaaatcat ttcggcagta catattgaag
     1981 cgtatatagg acgagtaact gtttgtgcag aagccattgc gattggtagt gcagtttcga
     2041 atggacaaaa ggattttgac acgattgtag ctgttagaca cccttattct gacgaagtag
     2101 atagaagtat tcgagtggta agtccttgtg gtatgtgtag ggagttgatt tcagactatg
     2161 caccagattg ttttgtgtta atagaaatga atggcaagtt agtcaaaact acgattgaag
     2221 aactcattcc actcaaatat acccgaaatt aaggatcttt ttccctctgc caaaaattat
     2281 ggggacatca tgaagcccct tgagggatcc gcccctctcc ctcccccccc cctaacgtta
     2341 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca
     2401 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca
     2461 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg
     2521 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc
     2581 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata
     2641 cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag
     2701 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc
     2761 attgtatggg atctgatctg gggcctcggt acacatgctt tacatgtgtt tagtcgaggt
     2821 taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat
     2881 gataatatgg ccacaaccat gtgcgataac atggtgagca agggcgagga gctgttcacc
     2941 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     3001 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     3061 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     3121 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     3181 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     3241 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     3301 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     3361 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     3421 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     3481 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     3541 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     3601 actctcggca tggacgagct gtacaagaag gagaagagtg cttgtcctaa agatccagcc
     3661 aaacctccgg ccaaggcaca agttgtggga tggccaccgg tgagatcata ccggaagaac
     3721 gtgatggttt cctgccaaaa atcaagcggt ggcccggagg cggcggcgtt cgtgaaggta
     3781 tcaatggacg gagcaccgta cttgaggaaa atcgatttga ggatgtataa aggagctggt
     3841 gcaggcgctg gagcgggtgc cgatatccaa ttgttgttgt taacttgttt attgcagctt
     3901 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3961 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttagcgctcg gtcgttcggc
     4021 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4081 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4141 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4201 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4261 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4321 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4381 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4441 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4501 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4561 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4621 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4681 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4741 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4801 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4861 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagcccct
     4921 gcgcaacgtg gcaggaagcg cgcgctggca aatgctaaac cactgcagtg gttaccagtg
     4981 cttgatcagt gaggcaccga tctcagcgat ctgcctattt cgttcgtcca tagtggcctg
     5041 actccccgtc gtgtagatca ctacgattcg tgagggctta ccatcaggcc ccagcgcagc
     5101 aatgatgccg cgagagccgc gttcaccggc ccccgatttg tcagcaatga accagccagc
     5161 agggagggcc gagcgaagaa gtggtcctgc tactttgtcc gcctccatcc agtctatgag
     5221 ctgctgtcgt gatgctagag taagaagttc gccagtgagt agtttccgaa gagttgtggc
     5281 cattgctact ggcatcgtgg tatcacgctc gtcgttcggt atggcttcgt tcaactctgg
     5341 ttcccagcgg tcaagccggg tcacatgatc acccatatta tgaagaaatg cagtcagctc
     5401 cttagggcct ccgatcgttg tcagaagtaa gttggccgcg gtgttgtcgc tcatggtaat
     5461 ggcagcacta cacaattctc ttaccgtcat gccatccgta agatgctttt ccgtgaccgg
     5521 cgagtactca accaagtcgt tttgtgagta gtgtatacgg cgaccaagct gctcttgccc
     5581 ggcgtctata cgggacaaca ccgcgccaca tagcagtact ttgaaagtgc tcatcatcgg
     5641 gaatcgttct tcggggcgga aagactcaag gatcttgccg ctattgagat ccagttcgat
     5701 atagcccact cttgcaccca gttgatcttc agcatctttt actttcacca gcgtttcggg
     5761 gtgtgcaaaa acaggcaagc aaaatgccgc aaagaaggga atgagtgcga cacgaaaatg
     5821 ttggatgctc atactcgtcc tttttcaata ttattgaagc atttatcagg gttactagta
     5881 cgtctctcaa ggataagtaa gtaatattaa ggtacgggag gtattggaca ggccgcaata
     5941 aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg atagtactaa
     6001 catacgctct ccatcaaaac aaaacgaaac aaaacaaact agcaaaatag gctgtcccca
     6061 gtgcaagtgc aggtgccaga acatttctct ggcctaacca ggctccccag caggcagaag
     6121 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     6181 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     6241 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     6301 cttttttgga ggcctaggct tttgcaaaaa gctttgcaaa gatggataaa gttttaaaca
     6361 gagaggaatc tttgcagcta atggaccttc taggtcttga aaggagtggg aattggctcc
     6421 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     6481 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     6541 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     6601 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt
     6661 ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc
     6721 acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt
     6781 tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg
     6841 cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat
     6901 aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat
     6961 agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc
     7021 ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg
     7081 ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc
     7141 gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg
     7201 tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg
     7261 cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca
     7321 gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc
     7381 tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt
     7441 ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc
     7501 ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt
     7561 caaagttttt ttcttccatt tcaggtgtcg tga
//
