LOCUS       sequence-478763-        7469 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Exrpession of Rice TIR1 F74->G, Rice ARF fragment and blaticidin 
            resistance by read through transaction after integration int the 
            PFR1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7469)
  AUTHORS   Alexandra Klein, Alice McDowell, Christian R S Reis, Rodrigo Pontes 
            de Lima, Danielle M N Moura, Janaina de Freitas Nascimento, Osvaldo 
            P de Melo Neto, Mark Carrington
  TITLE     An auxin-inducible degron system for trypanosomes
  JOURNAL   bioRxiv 2025.06.17.660065
REFERENCE   2  (bases 1 to 7469)
  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: "bioRxiv 
            2025.06.17.660065"
COMMENT     Sequence Label: p5179
FEATURES             Location/Qualifiers
     source          1..7469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             443..2167
                     /codon_start=1
                     /product="rice transport inhibitor response 1 protein, 
                     mutated for use in the AID2 system of inducible degradation
                     (Yesbolatova et al., 2020)"
                     /label=OsTIR1(F74G)
                     /note="F-box protein that mediates auxin-dependent 
                     interaction of a ubiquitin ligase with an auxin-inducible 
                     degron (Tan et al., 2007)"
                     /translation="MTYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIERLSRRGVFV
                     GNCYAVRAGRVAARFPNVRALTVKGKPHGADFNLVPPDWGGYAGPWIEAAARGCHGLEE
                     LRMKRMVVSDESLELLARSFPRFRALVLISCEGFSTDGLAAVASHCKLLRELDLQENEV
                     EDRGPRWLSCFPDSCTSLVSLNFACIKGEVNAGSLERLVSRSPNLRSLRLNRSVSVDTL
                     AKILLRTPNLEDLGTGNLTDDFQTESYFKLTSALEKCKMLRSLSGFWDASPVCLSFIYP
                     LCAQLTGLNLSYAPTLDASDLTKMISRCVKLQRLWVLDCISDKGLQVVASSCKDLQELR
                     VFPSDFYVAGYSAVTEEGLVAVSLGCPKLNSLLYFCHQMTNAALVTVAKNCPNFTRFRL
                     CILEPGKPDVVTSQPLDEGFGAIVRECKGLQRLSISGLLTDKVFMYIGKYAKQLEMLSI
                     AFAGDSDKGMMHVMNGCKNLRKLEIRDSPFGDAALLGNFARYETMRSLWMSSCNVTLKG
                     CQVLASKMPMLNVEVINERDGSNEMEENHGDLPKVEKLYVYRTTAGARDDAPNFVKIL"
     promoter        complement(4594..4612)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4595..4612)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4630..4646)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4630..4646)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4643..4665)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4654..4670
                     /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(4678..4684,4685..4702,4703..4708))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4723..4744
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4861..4878)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5032..5620)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5112..5131)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5791..6582,6583..6651))
                     /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     6414..6433
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6652..6756)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6834..7289)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6971..6992)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7183..7202
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7415..7437
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7429..7446
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7430..7446
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(7453..7469,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(7453..7469,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctta attaatgtgg ccgcaattat tatgtaaatt
       61 gagtgcaact agttatttga tgccaatgtc ttgtgcagtc ttttcttatt cttgtactag
      121 atgtaataaa ggcccaactg ggtcggtata tgtaggtcac catgatgcgt tttcgagcat
      181 ccgctctgtt attgtgccaa accgacggct tccggcacca ccacttgcct gtgtagcttt
      241 aacattgcac tgatgctcac acttttcttc atcatcttgt acgcgtccgg caaacagttt
      301 ttcgcaatag cttgtaggaa agcaaggaat aagatctaga aacatcacgt aagaacttca
      361 agcaccagac agaaatcaca caactttcaa cacaagaaat cgattatcct taataaaaga
      421 gtgctaccaa gcttccgcaa caatgaccta cttcccggag gaggtggtgg agcacatctt
      481 ctccttcctc ccggctcagc gtgaccgtaa cacggtgtcc ctcgtgtgca aggtctggta
      541 cgagattgag cgcctcagcc gccgtggagt gttcgtgggt aactgctacg ctgtgcgtgc
      601 tggacgtgtc gccgctcgtt tccccaacgt gcgtgctctc accgtgaaag gtaagcccca
      661 cggagctgac ttcaacctcg tccccccgga ctggggaggc tacgccggcc cgtggattga
      721 ggcagctgca cgcggctgcc acggtctcga ggagctccgt atgaaacgca tggtcgtctc
      781 cgacgagtcc ctcgagctcc tcgcccgtag cttcccgcgc ttccgtgccc tcgtgctcat
      841 ttcctgcgag ggcttcagca cagacggact cgccgctgtc gcaagccact gcaagctcct
      901 ccgtgagctc gacctccagg agaacgaggt ggaggaccgt ggtccgcgct ggctcagctg
      961 cttccccgac agctgcacca gcctcgtctc cctcaacttc gcctgcatca aaggagaggt
     1021 caacgctggt tccctcgagc gcctcgtgtc ccgcagcccg aacctccgtt ccctccgtct
     1081 caaccgtagc gtgtccgtcg acaccctcgc aaaaatcctc ctccgtacgc ccaacctcga
     1141 ggacctcggc acaggaaacc tcaccgacga cttccagacg gagtcctatt tcaaactcac
     1201 cagcgctctc gagaagtgca aaatgctccg ttccctcagc ggcttctggg acgcttcccc
     1261 ggtgtgcctc tccttcatct accccctctg cgctcagctc accggcctca acctctccta
     1321 cgctcccaca ctcgacgcat ccgacctcac caagatgatc tcccgttgcg tgaaactcca
     1381 gcgcctctgg gtgctcgact gcattagcga caaaggcctc caggtcgtgg ctagcagctg
     1441 caaagacctc caggagctcc gtgtgttccc ctccgacttc tatgtggccg gatattccgc
     1501 cgtcacagag gagggtctcg tggcagtctc cctcggatgc cccaagctca actccctcct
     1561 ctacttctgc caccagatga ccaacgccgc cctcgtcaca gtcgctaaga actgccccaa
     1621 cttcacacgc ttccgcctct gcattctcga gccgggtaag ccggacgtcg tgacctccca
     1681 gccgctcgac gagggtttcg gcgccatcgt gcgtgagtgc aagggactcc agcgcctctc
     1741 catttccggc ctcctcaccg acaaagtgtt catgtacatc ggaaagtacg ccaaacagct
     1801 cgagatgctc tccattgcat tcgctggaga cagcgacaag ggcatgatgc acgtgatgaa
     1861 cggctgcaaa aacctccgca agctcgagat ccgcgacagc ccgttcggcg acgcagcact
     1921 cctcggtaac ttcgcacgct acgagacaat gcgtagcctc tggatgtcct cctgcaacgt
     1981 gacactcaag ggatgccagg tcctcgcctc caaaatgccc atgctcaacg tggaggtgat
     2041 caacgagcgt gacggcagca acgagatgga ggagaaccac ggtgacctcc ccaaagtcga
     2101 gaagctctac gtgtaccgca ccaccgcagg cgcacgcgac gacgccccga acttcgtgaa
     2161 gattctcggt tccggatctt ccgaggtcca caccaaccag gacccgctcg actagtgtgg
     2221 ccgcaattat tatgtaaatt gagtgcaact agttatttga tgccaatgtc ttgtgcagtc
     2281 ttttcttatt cttgtactag atgtaataaa ggcccaactg ggtcggtata tgtaggtcac
     2341 catgatgcgt tttcgagcat ccgctctgtt attgtgccaa accgacggct tccggcacca
     2401 ccacttgcct gtgtagcttt aacattgcac tgatgctcac acttttcttc atcatcttgt
     2461 acgcgtccgg caaacagttt ttcgcaatag cttgtaggaa agcaaggaat aagatctaga
     2521 aacatcacgt aagaacttca agcaccagac agaaatcaca caactttcaa cacaagaaat
     2581 cgattatcct taataaaaga gtgctaccgg atccccgcaa caatgggcac agaggtgcac
     2641 accaatcagg accctctgga tggaaccggc accggcacaa cactgacaag cggacagagg
     2701 gaccaccgcg acgtggaaaa tgaactgagc agcgccgcct tcagcagcca gtcttttggc
     2761 gtgcccgaca tgagcttcaa gcctggctgt agctctgacg tggccgtgac agatgccgga
     2821 atgccttctc aaggcctgtg gaacaatcag acccagcgga tgcggacctt caccaaggtt
     2881 cagaagagag gcagcgtcgg ccggtccatc gacatcacca gatacagaga ctacgacgag
     2941 ctgcggcacg acctggcctg catgtttgga atccaaggcc agctggaaga tccctaccgg
     3001 atggactgga agctggtgta cgtggaccac gagaacgaca tcctgctcgt gggagatgac
     3061 ccctgggaag agtttgtggg ctgcgtgaag tccatcaaga tcctgtctgc cgccgaggtg
     3121 cagcagatga gtctggatgg cgatcttggc ggagtgcctc ctcagacaca ggcttgtagc
     3181 gccagcgacg atgccaatgc ttggagaggc aagctgagaa tcctgcagag caccgtgcct
     3241 cgggctagag atcctcctga cctggacaac tgatgtggcc gcaattatta tgtaaattga
     3301 gtgcaactag ttatttgatg ccaatgtctt gtgcagtctt ttcttattct tgtactagat
     3361 gtaataaagg cccaactggg tcggtatatg taggtcacca tgatgcgttt tcgagcatcc
     3421 gctctgttat tgtgccaaac cgacggcttc cggcaccacc acttgcctgt gtagctttaa
     3481 cattgcactg atgctcacac ttttcttcat catcttgtac gcgtccggca aacagttttt
     3541 cgcaatagct tgtaggaaag caaggaataa gatctagaaa catcacgtaa gaacttcaag
     3601 caccagacag aaatcacaca actttcaaca caagaaatcg attatcctta ataaaagagt
     3661 gctacccata tgcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca
     3721 atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac
     3781 ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc
     3841 gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc
     3901 ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat
     3961 ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg
     4021 attcgtgaat tgctgccctc tggttatgtg tgggagggct aattcgaatg tggccgcaat
     4081 tattatgtag atttttttgt ttgtagatgt ttttttctta tgaaccaacc cattcttcta
     4141 gaagttttat catagtttct actttttgag ttagtgatct ttgcgagatt gttacttgtg
     4201 ggtggtcgtg ctgtttgatg gaagtgtaga aaatgcttct tgttcacttc tttctgatgt
     4261 atttttaggt gataatctcc acttttttct gttaatggtt tgggttatgc tttttatgtt
     4321 tttttttctt ttttacctga ctgtattcct ttatttcatt ctttttttgt gtggtaaagg
     4381 ggctttatgg acgaaggttt ggatgatatt gctgaaaatg atgtagtggt tgacgttgtg
     4441 agtttgattg cgcatcaagg ccgcgttggt tttgcttctt atagcagcgg tctttgtgtt
     4501 gtgcgctgtt cagagtcgtt tgcgtcaagg tttgatggcg atgttccaat taactcaaca
     4561 ttaattaagc ggccgcctgc atagcttgag tattctatag tgtcacctaa atagcttggc
     4621 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4681 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4741 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4801 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4861 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4921 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4981 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5041 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5101 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5161 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5221 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5281 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5341 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5401 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5461 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5521 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5581 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5641 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5701 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5761 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5821 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5881 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5941 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6001 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6061 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6121 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6181 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6241 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6301 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6361 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6421 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6481 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6541 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6601 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6661 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6721 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6781 tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt
     6841 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     6901 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     6961 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     7021 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     7081 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     7141 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     7201 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     7261 cgcgcttaat gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg
     7321 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     7381 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     7441 gccagtgaat tgtaatacga ctcactata
//
