LOCUS       sequence-478802-        6139 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 6139)
  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 6139)
  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-puro-3Ty:OsAID:3Ty (p5229)
FEATURES             Location/Qualifiers
     source          1..6139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(3264..3282)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3265..3282)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3300..3316)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3300..3316)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3313..3335)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3324..3340
                     /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(3348..3354,3355..3372,3373..3378))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3393..3414
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3531..3548)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3702..4290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3782..3801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4461..5252,5253..5321))
                     /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     5084..5103
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5322..5426)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5504..5959)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5641..5662)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5853..5872
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6085..6107
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6099..6116
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6100..6116
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6123..6139,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6123..6139,1..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 atgactgaat
      181 acaagccaac ggttcgtctt gctacgcgag acgatgtgcc gcgggccgta cggacgctgg
      241 ctgctgcgtt cgctgattat cccgccaccc gacatacggt tgacccagat cgacacattg
      301 agcgagtcac agaacttcaa gagttattct taacacgggt gggcttggac attggtaagg
      361 tttgggttgc ggacgatggc gcggctgttg cggtgtggac gacgccagag tcagtcgaag
      421 cgggcgcagt gtttgctgag atcggtcccc gaatggcgga gcttagcggg tcatgcctcg
      481 ctgcacaaca gcagatggaa ggtctgttgg caccacatag acctaaggag ccggcttggt
      541 tcttagcgac agttggcgtc agtccggacc accaaggtaa ggggctgggt tcagcggttg
      601 ttcttccagg agtagaggct gcggaaaggg caggtgtgcc ggcgttcctt gaaacgtctg
      661 caccacgtaa ccttcctttt tacgagagat tggggtttac agtaacagca gatgttgaag
      721 tgcctgaagg gccgaggaca tggtgcatga cacgtaagcc gggagcctaa ccatggtgcc
      781 catttagttg gcctttccct tgtctcgtgt cttttccgtg gaaaggttcc cggagtaatc
      841 tgatggcaca gcagggaggt gcgcctgcag gttggttagg aaggggggat gatgtaaaag
      901 aagaaaatgg ggggattcga gccccacgcg ttgctcaagc tgtgtagcgc aggcgtttcc
      961 ttacatattt ctctaacagg cacggaagcc taacaaatac acttggctta tttttttgcc
     1021 ccctcatgtc ttgtacaaat atttgcgata gcttagctat cagccacatt aatcaaacaa
     1081 gtataccaac aagcccgaaa acagaaactc aactgcaacg ggtacctatg ggttctggta
     1141 gtggttccgg ttccggttct gaggtacaca caaaccagga cccactggac ggctccgagg
     1201 tgcataccaa tcaagatccg ctggacggct ctgaagttca caccaatcaa gatcccttgg
     1261 ataagcttat ggccggcctg ggcttcgatg agacagagct gagacttgga cttcccggcg
     1321 ctggcgaact ggccgctaga tcttctggca agagaggctt cgccgagaca atcgacctga
     1381 agctgaagct gcagcctgct gctccagctg ctgtgtctgg cgaagaaggc gcccaagagg
     1441 acaaagaaga tgccgacgca gccgccgctg ccgccgatga gaaaatgagc atgaagagaa
     1501 gcgccagcca gagcagcgtg gtcacagctg aacccgatcc tgacaagccc agagcaccta
     1561 aggctcaggt tgtcggatgg cctcctgtgc ggagcttcag aaagaatgtg ctggccgaga
     1621 agtgcaaggc tgccgctctg gttaaggttt ccatggatgg cgccccttac ctgcggaaga
     1681 tcgacgtggc catgtacaag agctaccccg agctgagcat ggccttccag aatatgttca
     1741 ccagcttcac catcggcaag tgcggctctc accagcagct gaaagagagc aacaagctgc
     1801 gggacgacct ggaatacgtg cccacctacg aggacaagga cggcgattgg atgcttgtgg
     1861 gagatgtgcc ctgggaaatg ttcgtggaaa gctgcaagcg gctgcggatc atgaagggct
     1921 ctgaggccat tggactggcc cctagagccg tggaaaagtg caagtctgga tccgaggtac
     1981 atacaaatca ggaccccctc gatggtagtg aagtccatac aaatcaggac ccgctggatg
     2041 gctcagaggt tcatacgaac caagatcctc ttgatggatc aggatctgga tcaggatcgg
     2101 gtagttaaga gctccgctgc gcttaaatgt ctttcattat aatcaatgta taacctttat
     2161 gtagtatttc aatctatgcc gctgtgtacg tgcactgcgg tgcctatcct tcggcattag
     2221 agagtcactg tttgtgtaga tcgtagctgc atgtctgaca tctggttcat tagtgccgtt
     2281 ttgagtctgc actgttgtgc aacttgatat tcatcactgc acaccaaccg tgtgtttcct
     2341 cctttgggtg caccctttaa tccttgtctt ctcctttttt gtctctttcc ccctcgaaaa
     2401 ggtgtcaaac tactgccgca taaactacgg ttatcccaaa tttaagagaa agcaataaag
     2461 catcagctag catgactgaa tacaagccaa cggttcgtct tgctacgcga gacgatgtgc
     2521 cgcgggccgt acggacgctg gctgctgcgt tcgctgatta tcccgccacc cgacatacgg
     2581 ttgacccaga tcgacacatt gagcgagtca cagaacttca agagttattc ttaacacggg
     2641 tgggcttgga cattggtaag gtttgggttg cggacgatgg cgcggctgtt gcggtgtgga
     2701 cgacgccaga gtcagtcgaa gcgggcgcag tgtttgctga gatcggtccc cgaatggcgg
     2761 agcttagcgg gtcacgcctc gctgcacaac agcagatgga aggtctgttg gcaccacata
     2821 gacctaagga gccggcttgg ttcttagcga cagttggcgt cagtccggac caccaaggta
     2881 aggggctggg ttcagcggtt gttcttccag gagtagaggc tgcggaaagg gcaggtgtgc
     2941 cggcgttcct tgaaacgtct gcaccacgta accttccttt ttacgagaga ttggggttta
     3001 cagtaacagc agatgttgaa gtgcctgaag ggccgaggac atggtgcatg acacgtaagc
     3061 cgggagccta actcgagtgc ccatttagtt ggcttttccc ttgtctcgtg tcttttccgt
     3121 ggaaaggttc ccggagtaat ctgatggcac agcagggagg tgcgcctgca ggttggttag
     3181 gaagggggga tgatgtaaaa gaagaaaatg gggggattcg agccccgcac aggtctctca
     3241 aattggatgc atagcttgag tattctatag tgtcacctaa atagcttggc gtaatcatgg
     3301 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     3361 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     3421 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3481 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3541 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3601 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3661 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3721 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3781 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3841 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3901 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3961 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4021 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4081 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4141 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4201 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4261 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4321 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4381 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4441 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4501 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataat tacgatacgg
     4561 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4621 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4681 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4741 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4801 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4861 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4921 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4981 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5041 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5101 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5161 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5221 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5281 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5341 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5401 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgatgcggtg
     5461 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat
     5521 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     5581 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5641 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5701 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5761 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     5821 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     5881 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcacttaat
     5941 gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     6001 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     6061 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat
     6121 tgtaatacga ctcactata
//
