LOCUS       sequence-478762-        6819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pPOTv7 based plasmid template for addition of HA-epitope tagged Rice
            Auxin Inducible Degradation (AID) domain to genes in trypanosomes 
            with selection using hygromycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6819)
  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 6819)
  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: pPOTv7-hyg-AtAID:HA (p5339)
FEATURES             Location/Qualifiers
     source          1..6819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             171..1196
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             171..1196
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1605..2288
                     /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"
     CDS             2304..2330
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2726..3751
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2726..3751
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(3944..3962)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3945..3962)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3980..3996)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3980..3996)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3993..4015)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4004..4020
                     /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(4028..4034,4035..4052,4053..4058))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4073..4094
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4211..4228)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4382..4970)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4462..4481)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5141..5932,5933..6001))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVIIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5764..5783
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6002..6106)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6184..6639)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6321..6342)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6533..6552
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6765..6787
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6779..6796
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6780..6796
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6803..6819,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6803..6819,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgtata atgcagacct gctgcaagct aggggcacag caaggtcttc
       61 tgaaattcat gttttttttt tttttactct gcattgcagt ctccgctctt atttagtttt
      121 gctttacgta aggtctcgtt gctgccataa aataagctct agtggaattc atgaaaaagc
      181 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
      241 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
      301 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
      361 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattta
      421 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
      481 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggcaatggat gcgatcgctg
      541 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
      601 acactacatg gcgtgatttt atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
      661 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
      721 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
      781 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
      841 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
      901 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgcct cggctccggg
      961 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     1021 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     1081 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     1141 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagccat
     1201 gggagctccg ctgcgcttaa atgtctttca ttataatcaa tgtataacct ttatgtagta
     1261 tttcaatcta tgccgctgtg tacgtgcact gcggtgccta tccttcggca ttagagagtc
     1321 actgtttgtg tagatcgtag ctgcatgtct gacatctggt tcattagtgc cgttttgagt
     1381 ctgcactgtt gtgcaacttg atattcatca ctgcacacca accgtgtgtt tcctcctttg
     1441 ggtgcaccct ttaatccttg tcttctcctt ttttgtctct ttccccctcg aaaaggtgtc
     1501 aaactactgc cgcataaact acggttatcc caaatttaag agaaagcaat aaagcatcag
     1561 gtaccgcaac aatgggttct ggtagtggtt ccggttccgg ttctatgggc agtgtcgagc
     1621 tgaatctgag ggagactgag ctgtgtcttg gtcttcccgg tggagataca gtggctccgg
     1681 taaccggaaa caagagaggg ttctcagaga cggttgatct gaagctaaat ctgaataatg
     1741 agcctgcaaa caaggaagga tctacgactc atgacgtcgt gacttttgat tccaaggaga
     1801 agagtgcttg tcctaaagat ccagccaaac ctccggccaa ggcacaagtt gtgggatggc
     1861 caccggtgag atcataccgg aagaacgtga tggtttcctg ccaaaaatca agcggtggcc
     1921 cggaggcggc ggcgttcgtg aaggtatcaa tggacggagc accgtacttg aggaaaatcg
     1981 atttgaggat gtataaaagc tacgatgagc tttctaatgc tttgtccaac atgttcagct
     2041 cttttactat gggcaaacat ggaggagaag aaggaatgat agacttcatg aatgagagga
     2101 aattgatgga tttggtgaat agctgggact atgttccctc ttatgaagac aaagacggtg
     2161 attggatgct cgtcggcgac gttccttggc caatgttcgt cgatacatgc aagcgtttac
     2221 gtctcatgaa aggatcggat gccattggtc tcgctccgag ggcgatggag aagtgcaaga
     2281 gcagagctgg ttccggatct tcctatccct atgatgtgcc agattacgct ggatccggat
     2341 ctggatcagg atcgggtagt taagagctct agaaagtgtg acaacgtcgc accatgtgta
     2401 ggttttcatt tatgttcttt ctttcttttt tttgtgaatt tgttttctgt ctcaaatgtt
     2461 tttaattcgc ttgggaccta tgtttttctt gtttttttgc tcaccctttg tgtaggaggc
     2521 accctgtcac gtctgtggtt gcgtgtatgc cttccttccc cttattcgct tcttcctgtc
     2581 gtgtcacacc tctttctctc tctccctttc cgccttttct ttcaatcttg ttttctcgac
     2641 cagccctact agaggagaaa gaatagtaac cctttcatca aagaaaatag ttcaaacgaa
     2701 ttcataagct tccgcaacag ctagcatgaa aaagcctgaa ctcaccgcga cgtctgtcga
     2761 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     2821 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     2881 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     2941 cccgattccg gaagtgcttg acattgggga atttagcgag agcctgacct attgcatctc
     3001 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     3061 gcagccggtc gcggaggcaa tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     3121 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg attttatatg
     3181 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     3241 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     3301 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     3361 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     3421 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     3481 gcatccggag cttgcaggat cgcctcggct ccgggcgtat atgctccgca ttggtcttga
     3541 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     3601 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     3661 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     3721 ccccagcact cgtccgaggg caaaggaata gctcgagtgc ccatttagtt ggcttttccc
     3781 ttgtctcgtg tcttttccgt ggaaaggttc ccggagtaat ctgatggcac agcagggagg
     3841 tgcgcctgca ggttggttag gaagggggga tgatgtaaaa gaagaaaatg gggggattcg
     3901 agccccgcac aggtctctca aattggatgc atagcttgag tattctatag tgtcacctaa
     3961 atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4021 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4081 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4141 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4201 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4261 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4321 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4381 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4441 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4501 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4561 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4621 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4681 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4741 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4801 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4861 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4921 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4981 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5041 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5101 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5161 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5221 tgtagataat tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5281 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5341 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5401 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5461 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5521 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5581 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5641 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5701 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5761 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5821 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5881 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5941 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6001 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6061 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6121 aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     6181 aggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     6241 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     6301 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     6361 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     6421 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     6481 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     6541 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     6601 ccacacccgc cgcacttaat gcgccgctac agggcgcgtc cattcgccat tcaggctgcg
     6661 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     6721 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     6781 taaaacgacg gccagtgaat tgtaatacga ctcactata
//
