LOCUS       sequence-478765-        9209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Rice Cullin, Rice SKP1, Rice Rbx1, and G418 resistance
            by read through transcription after integration into the tubulin 
            locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9209)
  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 9209)
  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: p4919
FEATURES             Location/Qualifiers
     source          1..9209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4852..5646
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4906..4925)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5516..5535
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        complement(6334..6352)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(6335..6352)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(6370..6386)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6370..6386)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6383..6405)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6394..6410
                     /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(6418..6424,6425..6442,6443..6448))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6463..6484
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6601..6618)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6772..7360)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6852..6871)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7531..8322,8323..8391))
                     /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     8154..8173
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8392..8496)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8574..9029)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8711..8732)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8923..8942
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9155..9177
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     9169..9186
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     9170..9186
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(9193..9209,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(9193..9209,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggccgcggga ttttaattaa
       61 tagaaagtgt gacaacgtcg caccatgtgt aggttttcat ttatgttctt tctttctttt
      121 ttttgtgaat ttgttttctg tctcaaatgt ttttaattcg cttgggacct atgtttttct
      181 tgtttttttg ctcacccttt gtgtaggagg caccctgtca cgtctgtggt tgcgtgtatg
      241 ccttccttcc ccttattcgc ttcttcctgt cgtgtcacac ctctttctct ctctcccttt
      301 ccgccttttc tttcaatctt gttttctcga ccagccctac tagaggagaa agaacagtaa
      361 ccctttcatc aaagaaaata gttcaaacga attcataaag cttccgcaac aatggcaacg
      421 cacgagcgca agacgattga cctcgagcaa ggatgggagt tcatgcaaaa gggaattacg
      481 aagctcaaga acattctcga gggaaagccc gagccccaat tcagcagcga ggactacatg
      541 atgctctaca cgacgattta caacatgtgc acgcaaaagc ccccccacga ctacagccaa
      601 caactctacg agaagtaccg cgagagcttc gaggagtaca ttacgagcat ggtgctcccc
      661 agcctccgcg agaagcacga cgagttcatg ctccgcgagc tcgtgaagcg ctggagcaac
      721 cacaaggtga tggtgcgctg gctcagccgc ttcttccact acctcgaccg ctacttcatt
      781 agccgccgca gcctccccca actcagcgag gtgggactca gctgcttccg cgacctcgtg
      841 taccaagaga ttaagggaaa ggtgaagagc gcagtgatta gcctcattga ccaagagcgc
      901 gagggagagc aaattgaccg cgcactcctc aagaacgtgc tcgacatttt cgtggagatt
      961 ggactcacga gcatggacta ctacgagaac gacttcgagg acttcctcct caaggacacg
     1021 gcagactact acagcattaa ggcacaaacg tggattctcg aggacagctg ccccgactac
     1081 atgctcaagg cagaggagtg cctcaagcgc gagaaggagc gcgtggcaca ctacctccac
     1141 agcagcagcg agcaaaagct cctcgagaag gtgcaacacg agctcctcac gcaatacgca
     1201 agccaactcc tcgagaagga gcacagcgga tgccacgcac tcctccgcga cgacaaggtg
     1261 gacgacctca gccgcatgta ccgcctcttc agccgcatta cgcgcggact cgagcccgtg
     1321 agccaaattt tcaagcaaca cgtgacgaac gagggaacgg cactcgtgaa gcaagcagag
     1381 gacgcagcaa gcaacaagaa gcccgagaag aaggagattg tgggactcca agagcaagtg
     1441 ttcgtgcgca agattattga gctccacgac aagtacgtgg catacgtgac ggactgcttc
     1501 caaggacaca cgctcttcca caaggcactc aaggaggcat tcgaggtgtt ctgcaacaag
     1561 ggagtgagcg gaagcagcag cgcagagctc ctcgcaacgt tctgcgacaa cattctcaag
     1621 aagggaggaa gcgagaagct cagcgacgag gcaattgagg acacgctcga gaaggtggtg
     1681 cgcctcctcg catacattag cgacaaggac ctcttcgcag agttctaccg caagaagctc
     1741 gcacgccgcc tcctcttcga caagagcgca aacgacgagc acgagcgcag cattctcacg
     1801 aagctcaagc aacaatgcgg aggacaattc acgagcaaga tggagggaat ggtgacggac
     1861 ctcacggtgg cacgcgacca ccaagcaaag ttcgaggagt tcattagcac gcacagcgag
     1921 ctcaaccccg gaattgcact cgcagtgacg gtgctcacga cgggattctg gcccagctac
     1981 aagagcttcg acattaacct ccccgcagag atggtgaagt gcgtggaggt gttcaaggag
     2041 ttctaccaaa cgcgcacgaa gcaccgcaag ctcacgtgga tttacagcct cggaacgtgc
     2101 aacattaacg caaagttcga ggcaaagacg attgagctca ttgtgacgac gtaccaagca
     2161 gcactcctcc tcctcttcaa cggagtggac cgcctcagct acagcgagat tgtgacgcaa
     2221 ctcaacctca gcgacgacga cgtggtgcgc ctcctccaca gcctcagctg cgcaaagtac
     2281 aagattctca gcaaggagcc caacaaccgc agcattagcc ccaacgacgt gttcgagttc
     2341 aacagcaagt tcacggacaa gctccgccgc ctcaagattc ccctcccccc cgtggacgag
     2401 aagaagaagg tggtggagga cgtggacaag gaccgccgct acgcaattga cgcaagcatt
     2461 gtgcgcatta tgaagagccg caaggtgctc ggacaccaac aactcgtgat ggagtgcgtg
     2521 gagcaactcg gacgcatgtt caagcccgac ttcaaggcaa ttaagaagcg cattgaggac
     2581 ctcattacgc gcgactacct cgagcgcgac aaggacaacc ccaacgtgta ccgctacctc
     2641 gcaggttccg gatcttccga ggtccacacc aaccaggacc cgctcgacta gtgtggccgc
     2701 aattattatg taaattgagt gcaactagtt atttgatgcc aatgtcttgt gcagtctttt
     2761 cttattcttg tactagatgt aataaaggcc caactgggtc ggtatatgta ggtcaccatg
     2821 atgcgttttc gagcatccgc tctgttattg tgccaaaccg acggcttccg gcaccaccac
     2881 ttgcctgtgt agctttaaca ttgcactgat gctcacactt ttcttcatca tcttgtacgc
     2941 gtccggcaaa cagtttttcg caatagcttg taggaaagca aggaataaga tctagaaaca
     3001 tcacgtaaga acttcaagca ccagacagaa atcacacaac tttcaacaca agaaatcgat
     3061 tatccttaat aaaagagtgc taccggatcc ccgcaacaat ggctgccgag ggcgagaaaa
     3121 agatgattac gctcaagagc tccgacggtg aggagttcga ggtggaggag gccgtcgcta
     3181 tggagagcca gaccatccgc cacatgattg aggacgactg cgcagacaac ggaatccccc
     3241 tccccaacgt caactccaag attctcagca aggtgatcga gtattgcaac aagcacgtcc
     3301 acgctgccgc cgctgccgca tccaaagcag ccgacgacgc tgcttccgcc gcagccgccg
     3361 tgcccccgcc gagcggcgag gacctcaaga actgggacgc tgacttcgtc aaggtcgacc
     3421 aggctacact cttcgacctc atcctcgctg caaactatct caacattaaa ggactcctcg
     3481 acctcacgtg ccagacggtg gcagacatga tcaagggaaa aacaccggag gagattcgta
     3541 agacgttcaa cattaagaac gacttcacac cggaggagga ggaggagatc cgtcgtgaga
     3601 accagtgggc attcgagggt tccggatctt ccgaggtcca caccaaccag gacccgctcg
     3661 actagtgtgg ccgcaattat tatgtaaatt gagtgcaact agttatttga tgccaatgtc
     3721 ttgtgcagtc ttttcttatt cttgtactag atgtaataaa ggcccaactg ggtcggtata
     3781 tgtaggtcac catgatgcgt tttcgagcat ccgctctgtt attgtgccaa accgacggct
     3841 tccggcacca ccacttgcct gtgtagcttt aacattgcac tgatgctcac acttttcttc
     3901 atcatcttgt acgcgtccgg caaacagttt ttcgcaatag cttgtaggaa agcaaggaat
     3961 aagatctaga aacatcacgt aagaacttca agcaccagac agaaatcaca caactttcaa
     4021 cacaagaaat cgattatcct taataaaaga gtgctacccg caacaatgga caagggagac
     4081 gtggcagtgg cagtgccccc cagcattgca ggagcaagca gcagcggagc aaagaaggga
     4141 aagcgcttcg agattaagaa gtggaacgca gtgagcctct gggcatggga cattgtggtg
     4201 gacaactgcg caatttgccg caaccacatt atggacctct gcattgagtg ccaagcaaac
     4261 caagcaagcg caacgagcga ggagtgcacg gtggcatggg gagtgtgcaa ccacgcattc
     4321 cacttccact gcattagccg ctggctcaag acgcgccaag tgtgccccct cgacaacagc
     4381 gagtgggagt tccaaaagta cggacacggt tccggatctt ccgaggtcca caccaaccag
     4441 gacccgctcg actagtgtgg ccgcaattat tatgtaaatt gagtgcaact agttatttga
     4501 tgccaatgtc ttgtgcagtc ttttcttatt cttgtactag atgtaataaa ggcccaactg
     4561 ggtcggtata tgtaggtcac catgatgcgt tttcgagcat ccgctctgtt attgtgccaa
     4621 accgacggct tccggcacca ccacttgcct gtgtagcttt aacattgcac tgatgctcac
     4681 acttttcttc atcatcttgt acgcgtccgg caaacagttt ttcgcaatag cttgtaggaa
     4741 agcaaggaat aagatctaga aacatcacgt aagaacttca agcaccagac agaaatcaca
     4801 caactttcaa cacaagaaat cgattatcct taataaaaga gtgctaccca tatgattgaa
     4861 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4921 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4981 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     5041 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     5101 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     5161 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     5221 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     5281 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     5341 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     5401 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     5461 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     5521 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     5581 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     5641 ttctgattcg aatagacgcg gacggggcat ttcccgttcg tcattagcag taggtaatga
     5701 agatgtttgt ttctcgtccc ctttctcctt cgtccttctg tcattttgtt cttttgtgtt
     5761 tatgttttgt tgttgttttc tttaattttt ttttttcttc cacgtttgtg tacatccgcg
     5821 cgccactcta ttcagagagc cacggatagt agaggaggtg ggaagggtat atgagggaca
     5881 cgcgtaccat gatgtgggat gtattggggt ccctgtctgt ccttacgtga ctatgtatga
     5941 accgtcacgt gtaagatgag ctagtgagat caacagtaca actcatcaac acgccttctt
     6001 ctcgttaaat gtacacaatc ttgatcctcc acctttatgg gtcccattgt ttgcctcttc
     6061 cgctgtgtgg agtgcgccta cacgcacttc tcacttcgta agtggtggtg gcgtaagtat
     6121 tgcctaatgt tgactctata ttctcctctc ctcaccccct cgcggtgctg atttctgaca
     6181 gatcttcaaa cactagttta agcaaaggac tattcatccg tttatattag caacagtagg
     6241 tactagcacc actaacaaca acaacaaagc acttctattt atttatcatg ttaattaaaa
     6301 tcactagtgc ggccgcctgc atagcttgag tattctatag tgtcacctaa atagcttggc
     6361 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     6421 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     6481 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6541 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     6601 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     6661 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     6721 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     6781 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     6841 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6901 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6961 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     7021 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     7081 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     7141 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     7201 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     7261 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     7321 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     7381 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     7441 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     7501 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     7561 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     7621 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     7681 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     7741 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     7801 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     7861 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     7921 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     7981 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     8041 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     8101 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     8161 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     8221 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     8281 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     8341 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     8401 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     8461 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     8521 tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt
     8581 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     8641 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     8701 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     8761 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     8821 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     8881 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     8941 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     9001 cgcgcttaat gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg
     9061 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     9121 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     9181 gccagtgaat tgtaatacga ctcactata
//
