LOCUS       sequence-355150-        5741 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Trypanosoma brucei PCR only tagging (pPOT) vector for endogenous 
            gene tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5741)
  AUTHORS   Paterou A, Saez Conde J, Tyc J, Sunter JD, Vaughan S, Gull K, Dean S
  TITLE     A comprehensive toolkit for protein localization and functional 
            analysis in trypanosomatids.
  JOURNAL   Open Biol. 2025 Apr;15(4):240361. doi: 10.1098/rsob.240361. Epub 
            2025 Apr 2.
  PUBMED    40169015
REFERENCE   2  (bases 1 to 5741)
  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: "Open 
            Biol."; date: "2025-04"; volume: "15(4):240361. doi"; pages: " 
            10.1098/rsob.240361. Epub 2025 Apr 2"
COMMENT     Sequence Label: pPOTv6-blast-3Ty:pHlourinM153R:3Ty
FEATURES             Location/Qualifiers
     source          1..5741
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2278..2673
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AMPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(2866..2884)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2867..2884)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2902..2918)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2902..2918)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2915..2937)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2926..2942
                     /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(2950..2956,2957..2974,2975..2980))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2995..3016
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3133..3150)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3304..3892)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3384..3403)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4063..4854,4855..4923))
                     /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     4686..4705
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4924..5028)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5106..5561)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5243..5264)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5455..5474
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5687..5709
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5701..5718
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5702..5718
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5725..5741,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5725..5741,1..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 atgcctttgt
      181 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct
      241 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg
      301 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg
      361 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca
      421 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga cctgcatcct gggatcaaag
      481 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct
      541 ctggttatgt gtgggagggc taaccatggt gcccatttag ttggcctttc ccttgtctcg
      601 tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga ggtgcgcctg
      661 caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt cgagccccac
      721 gcgttgctca agctgtgtag cgcaggcgtt tccttacata tttctctaac aggcacggaa
      781 gcctaacaaa tacacttggc ttattttttt gccccctcat gtcttgtaca aatatttgcg
      841 atagcttagc tatcagccac attaatcaaa caagtatacc aacaagcccg aaaacagaaa
      901 ctcaactgca acgggtacct atgggttctg gtagtggttc cggttccggt tctgaggtac
      961 acacaaacca ggacccactg gacggctccg aggtgcatac caatcaagat ccgctggacg
     1021 gctctgaagt tcacaccaat caagatccct tggataagct tatgtctaaa ggcgaagaat
     1081 tgttcactgg ggtagtacct attttggtag aattggacgg agacgttaat ggacacaaat
     1141 tctccgtgtc gggtgagggt gaaggcgacg caacttatgg taagttgacc ctgaaattca
     1201 tatgtaccac gggaaaattg ccagttccgt ggccgacgct cgtcactacg ttctcctacg
     1261 gagtacagtg tttttcacgg tatccagatc atatgaaacg gcatgatttc ttcaaatcag
     1321 cgatgccgga aggctatgtg caggagcgga ccattttctt caaggacgat ggtaattaca
     1381 agacccgagc agaagtcaaa ttcgaaggcg acaccttggt taatcggatc gaacttaaag
     1441 gtatcgattt caaagatgat ggtaacatac tcggccataa gctcgaatac aactataatg
     1501 agcaccttgt ctatatacga gctgacaaac aaaagaatgg aacaaaagcg attttccagg
     1561 tacatcataa tatcgaggat ggtggcgtcc agcttgcaga tcactatcag cagaatactc
     1621 ctatcggtga cggacctgta ttgcttcccg ataatcatta cttgcacact cagtcagcgc
     1681 tctccaaaga tccgaacgag aaacgagatc atatggttct tctggagttc gtcactgccg
     1741 ctggcattac tcatggaatg gacgaacttt acaagggatc cgaggtacat acaaatcagg
     1801 accccctcga tggtagtgaa gtccatacaa atcaggaccc gctggatggc tcagaggttc
     1861 atacgaacca agatcctctt gatggatcag gatctggatc aggatcgggt agttaagagc
     1921 tccgctgcgc ttaaatgtct ttcattataa tcaatgtata acctttatgt agtatttcaa
     1981 tctatgccgc tgtgtacgtg cactgcggtg cctatccttc ggcattagag agtcactgtt
     2041 tgtgtagatc gtagctgcat gtctgacatc tggttcatta gtgccgtttt gagtctgcac
     2101 tgttgtgcaa cttgatattc atcactgcac accaaccgtg tgtttcctcc tttgggtgca
     2161 ccctttaatc cttgtcttct ccttttttgt ctctttcccc ctcgaaaagg tgtcaaacta
     2221 ctgccgcata aactacggtt atcccaaatt taagagaaag caataaagca tcagctagcc
     2281 atgcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc
     2341 atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc
     2401 ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg
     2461 ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag
     2521 aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct
     2581 gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa
     2641 ttgctgccct ctggttatgt gtgggagggc taactcgagt gcccatttag ttggcttttc
     2701 ccttgtctcg tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga
     2761 ggtgcgcctg caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt
     2821 cgagccccgc acaggtctct caaattggat gcatagcttg agtattctat agtgtcacct
     2881 aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     2941 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3001 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3061 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3121 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3181 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3241 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3301 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3361 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3421 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3481 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3541 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3601 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3661 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3721 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3781 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3841 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3901 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3961 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4021 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4081 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4141 cgtgtagata attacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4201 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4261 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4321 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4381 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4441 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4501 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4561 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4621 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4681 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4741 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4801 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4861 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4921 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4981 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5041 aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     5101 tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5161 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5221 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5281 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     5341 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     5401 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     5461 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     5521 caccacaccc gccgcactta atgcgccgct acagggcgcg tccattcgcc attcaggctg
     5581 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     5641 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     5701 tgtaaaacga cggccagtga attgtaatac gactcactat a
//
