LOCUS       sequence-478812-        5935 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pPOTv7 based plasmid template for addition of a 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 5935)
  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 5935)
  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-puro-OsAID (p5109)
FEATURES             Location/Qualifiers
     source          1..5935
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(3060..3078)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3061..3078)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3096..3112)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3096..3112)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3109..3131)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3120..3136
                     /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(3144..3150,3151..3168,3169..3174))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3189..3210
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3327..3344)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3498..4086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3578..3597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4257..5048,5049..5117))
                     /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     4880..4899
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5118..5222)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5300..5755)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5437..5458)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5649..5668
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5881..5903
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5895..5912
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5896..5912
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5919..5935,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5919..5935,1..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 aagcttatgg ccggcctggg cttcgatgag acagagctga
     1201 gacttggact tcccggcgct ggcgaactgg ccgctagatc ttctggcaag agaggcttcg
     1261 ccgagacaat cgacctgaag ctgaagctgc agcctgctgc tccagctgct gtgtctggcg
     1321 aagaaggcgc ccaagaggac aaagaagatg ccgacgcagc cgccgctgcc gccgatgaga
     1381 aaatgagcat gaagagaagc gccagccaga gcagcgtggt cacagctgaa cccgatcctg
     1441 acaagcccag agcacctaag gctcaggttg tcggatggcc tcctgtgcgg agcttcagaa
     1501 agaatgtgct ggccgagaag tgcaaggctg ccgctctggt taaggtttcc atggatggcg
     1561 ccccttacct gcggaagatc gacgtggcca tgtacaagag ctaccccgag ctgagcatgg
     1621 ccttccagaa tatgttcacc agcttcacca tcggcaagtg cggctctcac cagcagctga
     1681 aagagagcaa caagctgcgg gacgacctgg aatacgtgcc cacctacgag gacaaggacg
     1741 gcgattggat gcttgtggga gatgtgccct gggaaatgtt cgtggaaagc tgcaagcggc
     1801 tgcggatcat gaagggctct gaggccattg gactggcccc tagagccgtg gaaaagtgca
     1861 agtctggatc cggatcagga tctggatcag gatcgggtag ttaagagctc cgctgcgctt
     1921 aaatgtcttt cattataatc aatgtataac ctttatgtag tatttcaatc tatgccgctg
     1981 tgtacgtgca ctgcggtgcc tatccttcgg cattagagag tcactgtttg tgtagatcgt
     2041 agctgcatgt ctgacatctg gttcattagt gccgttttga gtctgcactg ttgtgcaact
     2101 tgatattcat cactgcacac caaccgtgtg tttcctcctt tgggtgcacc ctttaatcct
     2161 tgtcttctcc ttttttgtct ctttccccct cgaaaaggtg tcaaactact gccgcataaa
     2221 ctacggttat cccaaattta agagaaagca ataaagcatc agctagcatg actgaataca
     2281 agccaacggt tcgtcttgct acgcgagacg atgtgccgcg ggccgtacgg acgctggctg
     2341 ctgcgttcgc tgattatccc gccacccgac atacggttga cccagatcga cacattgagc
     2401 gagtcacaga acttcaagag ttattcttaa cacgggtggg cttggacatt ggtaaggttt
     2461 gggttgcgga cgatggcgcg gctgttgcgg tgtggacgac gccagagtca gtcgaagcgg
     2521 gcgcagtgtt tgctgagatc ggtccccgaa tggcggagct tagcgggtca cgcctcgctg
     2581 cacaacagca gatggaaggt ctgttggcac cacatagacc taaggagccg gcttggttct
     2641 tagcgacagt tggcgtcagt ccggaccacc aaggtaaggg gctgggttca gcggttgttc
     2701 ttccaggagt agaggctgcg gaaagggcag gtgtgccggc gttccttgaa acgtctgcac
     2761 cacgtaacct tcctttttac gagagattgg ggtttacagt aacagcagat gttgaagtgc
     2821 ctgaagggcc gaggacatgg tgcatgacac gtaagccggg agcctaactc gagtgcccat
     2881 ttagttggct tttcccttgt ctcgtgtctt ttccgtggaa aggttcccgg agtaatctga
     2941 tggcacagca gggaggtgcg cctgcaggtt ggttaggaag gggggatgat gtaaaagaag
     3001 aaaatggggg gattcgagcc ccgcacaggt ctctcaaatt ggatgcatag cttgagtatt
     3061 ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3121 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3181 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3241 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3301 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3361 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3421 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3481 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3541 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3601 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3661 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3721 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3781 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3841 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3901 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3961 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4021 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4081 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4141 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4201 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4261 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4321 gcctgactcc ccgtcgtgta gataattacg atacgggagg gcttaccatc tggccccagt
     4381 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4441 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4501 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4561 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4621 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4681 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4741 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4801 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4861 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4921 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4981 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5041 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5101 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5161 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5221 cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag
     5281 gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     5341 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     5401 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     5461 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     5521 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     5581 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     5641 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     5701 acgctgcgcg taaccaccac acccgccgca cttaatgcgc cgctacaggg cgcgtccatt
     5761 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     5821 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     5881 cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca ctata
//
