LOCUS       sequence-478759-        6991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pPOTv6 based plasmid template for addition of Ty-epitope tagged Rice
            Auxin Inducible Degradation (AID) domain to genes in trypanosomes 
            with selection using puromycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6991)
  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 6991)
  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: pPOTv6-hyg-3Ty:OsAID:3Ty (p5230)
FEATURES             Location/Qualifiers
     source          1..6991
                     /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             2898..3923
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2898..3923
                     /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(4116..4134)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4117..4134)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4152..4168)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4152..4168)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4165..4187)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4176..4192
                     /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(4200..4206,4207..4224,4225..4230))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4245..4266
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4383..4400)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4554..5142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4634..4653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5313..6104,6105..6173))
                     /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     5936..5955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6174..6278)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6356..6811)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6493..6514)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6705..6724
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6937..6959
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6951..6968
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6952..6968
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6975..6991,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6975..6991,1..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 ggtgcccatt tagttggcct ttcccttgtc tcgtgtcttt tccgtggaaa ggttcccgga
     1261 gtaatctgat ggcacagcag ggaggtgcgc ctgcaggttg gttaggaagg ggggatgatg
     1321 taaaagaaga aaatgggggg attcgagccc cacgcgttgc tcaagctgtg tagcgcaggc
     1381 gtttccttac atatttctct aacaggcacg gaagcctaac aaatacactt ggcttatttt
     1441 tttgccccct catgtcttgt acaaatattt gcgatagctt agctatcagc cacattaatc
     1501 aaacaagtat accaacaagc ccgaaaacag aaactcaact gcaacgggta cctatgggtt
     1561 ctggtagtgg ttccggttcc ggttctgagg tacacacaaa ccaggaccca ctggacggct
     1621 ccgaggtgca taccaatcaa gatccgctgg acggctctga agttcacacc aatcaagatc
     1681 ccttggataa gcttatggcc ggcctgggct tcgatgagac agagctgaga cttggacttc
     1741 ccggcgctgg cgaactggcc gctagatctt ctggcaagag aggcttcgcc gagacaatcg
     1801 acctgaagct gaagctgcag cctgctgctc cagctgctgt gtctggcgaa gaaggcgccc
     1861 aagaggacaa agaagatgcc gacgcagccg ccgctgccgc cgatgagaaa atgagcatga
     1921 agagaagcgc cagccagagc agcgtggtca cagctgaacc cgatcctgac aagcccagag
     1981 cacctaaggc tcaggttgtc ggatggcctc ctgtgcggag cttcagaaag aatgtgctgg
     2041 ccgagaagtg caaggctgcc gctctggtta aggtttccat ggatggcgcc ccttacctgc
     2101 ggaagatcga cgtggccatg tacaagagct accccgagct gagcatggcc ttccagaata
     2161 tgttcaccag cttcaccatc ggcaagtgcg gctctcacca gcagctgaaa gagagcaaca
     2221 agctgcggga cgacctggaa tacgtgccca cctacgagga caaggacggc gattggatgc
     2281 ttgtgggaga tgtgccctgg gaaatgttcg tggaaagctg caagcggctg cggatcatga
     2341 agggctctga ggccattgga ctggccccta gagccgtgga aaagtgcaag tctggatccg
     2401 aggtacatac aaatcaggac cccctcgatg gtagtgaagt ccatacaaat caggacccgc
     2461 tggatggctc agaggttcat acgaaccaag atcctcttga tggatcagga tctggatcag
     2521 gatcgggtag ttaagagctc cgctgcgctt aaatgtcttt cattataatc aatgtataac
     2581 ctttatgtag tatttcaatc tatgccgctg tgtacgtgca ctgcggtgcc tatccttcgg
     2641 cattagagag tcactgtttg tgtagatcgt agctgcatgt ctgacatctg gttcattagt
     2701 gccgttttga gtctgcactg ttgtgcaact tgatattcat cactgcacac caaccgtgtg
     2761 tttcctcctt tgggtgcacc ctttaatcct tgtcttctcc ttttttgtct ctttccccct
     2821 cgaaaaggtg tcaaactact gccgcataaa ctacggttat cccaaattta agagaaagca
     2881 ataaagcatc agctagcatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     2941 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
     3001 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     3061 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     3121 cggaagtgct tgacattggg gaatttagcg agagcctgac ctattgcatc tcccgccgtg
     3181 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     3241 tcgcggaggc aatggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     3301 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgattttata tgcgcgattg
     3361 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     3421 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     3481 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     3541 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     3601 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     3661 agcttgcagg atcgcctcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     3721 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     3781 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     3841 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     3901 ctcgtccgag ggcaaaggaa tagctcgagt gcccatttag ttggcttttc ccttgtctcg
     3961 tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga ggtgcgcctg
     4021 caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt cgagccccgc
     4081 acaggtctct caaattggat gcatagcttg agtattctat agtgtcacct aaatagcttg
     4141 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4201 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4261 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4321 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4381 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4441 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4501 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4561 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4621 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4681 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4741 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4801 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4861 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4921 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4981 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5041 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5101 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5161 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5221 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5281 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5341 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5401 attacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5461 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5521 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5581 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5641 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5701 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5761 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5821 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5881 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5941 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6001 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6061 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6121 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6181 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6241 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6301 cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt
     6361 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     6421 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     6481 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     6541 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     6601 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     6661 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     6721 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     6781 gccgcactta atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt
     6841 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     6901 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     6961 cggccagtga attgtaatac gactcactat a
//
