LOCUS       sequence-355409-        6461 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 6461)
  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 6461)
  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:syPher2:3Ty
FEATURES             Location/Qualifiers
     source          1..6461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2998..3393
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AMPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(3586..3604)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3587..3604)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3622..3638)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3622..3638)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3635..3657)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3646..3662
                     /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(3670..3676,3677..3694,3695..3700))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3715..3736
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3853..3870)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4024..4612)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4104..4123)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4783..5574,5575..5643))
                     /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     5406..5425
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5644..5748)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5826..6281)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5963..5984)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6175..6194
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6407..6429
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6421..6438
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6422..6438
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6445..6461,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6445..6461,1..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 tatggaaatg gcgtctcagc
     1081 agggcgaaac catgtcagga cccttgcaca taggactcat acctaccgtt ggtccctact
     1141 tgctcccgca tataatccca atgctccacc agacctttcc aaaactggag atgtatctgc
     1201 acgaggctca gacacaccaa ctgcttgcac agctcgactc agggaaactt gactgtgtta
     1261 tactcgccct cgtcaaggaa tctgaggcat tcattgaggt gccgcttttt gatgaaccta
     1321 tgttgttggc catttacgaa gatcacccct gggctaaccg cgaatgcgtt ccgatggccg
     1381 atttggctgg tgagaagctg ctgatgcttg aggacggaca ctctttgcgg gatcaagcta
     1441 tgtccgcagg ctataactcc gataacgtgt atataatggc ggataagcag aagaacggta
     1501 taaaggctaa tttcaaaata cgtcataacg tcgaggacgg gtctgttcag ctcgcggatc
     1561 actaccagca aaacacgcca atcggggatg gaccagtatt gctgccagac aatcactatt
     1621 tgtcctttca gagtgtgctt tccaaggacc cgaacgaaaa gcgagatcat atggttcttt
     1681 tggagtttgt aaccgcggcg ggaatcactc tcggcatgga cgaactgtac aacgtagatg
     1741 gaggatctgg ggggaccggc tctaagggtg aggagctttt cactggggtt gtcccgatct
     1801 tggtcgagtt ggacggcgac gtcaacggac acaaattcag cgtctcgggg gagggcgaag
     1861 gggatgccac atatgggaag ctcacactca agttgatatg cactacgggg aagttgccag
     1921 ttccctggcc aacacttgtg accaccctgg gttacggttt gaaatgcttc gcacgatacc
     1981 cagaccatat gaagcaacac gacttcttta agtctgctat gcctgagggg tatgtgcagg
     2041 aaaggaccat cttctttaag gatgacggta actataagac ccgagcggag gtaaaatttg
     2101 agggcgatac gctggtaaat cggatagagt tgaagggtat cgatttcaaa gaggacggta
     2161 atatactcgg ccataagttg gagtataacg gtacggggtt ctgctttgaa gctggggcag
     2221 atgaagacac gcattttagg gccacttcac tcgaaactct gcggaacatg gttgcggtgg
     2281 gtagcgggat cacgcttctt ccagctcttg ccgttccgcc cgaacgaaag agggacggcg
     2341 tagtatattt gccgtgcatc aagccggaac ctcgtcgaac tatagggctc gtctatcggc
     2401 caggctctcc gttgcgctca cgatatgaac agctcgcgga agcgataagg gcgcgcatgg
     2461 acgggcattt tgacaaagtt ctcaaacagg cagtgggatc cgaggtacat acaaatcagg
     2521 accccctcga tggtagtgaa gtccatacaa atcaggaccc gctggatggc tcagaggttc
     2581 atacgaacca agatcctctt gatggatcag gatctggatc aggatcgggt agttaagagc
     2641 tccgctgcgc ttaaatgtct ttcattataa tcaatgtata acctttatgt agtatttcaa
     2701 tctatgccgc tgtgtacgtg cactgcggtg cctatccttc ggcattagag agtcactgtt
     2761 tgtgtagatc gtagctgcat gtctgacatc tggttcatta gtgccgtttt gagtctgcac
     2821 tgttgtgcaa cttgatattc atcactgcac accaaccgtg tgtttcctcc tttgggtgca
     2881 ccctttaatc cttgtcttct ccttttttgt ctctttcccc ctcgaaaagg tgtcaaacta
     2941 ctgccgcata aactacggtt atcccaaatt taagagaaag caataaagca tcagctagcc
     3001 atgcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc
     3061 atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc
     3121 ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg
     3181 ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag
     3241 aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct
     3301 gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa
     3361 ttgctgccct ctggttatgt gtgggagggc taactcgagt gcccatttag ttggcttttc
     3421 ccttgtctcg tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga
     3481 ggtgcgcctg caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt
     3541 cgagccccgc acaggtctct caaattggat gcatagcttg agtattctat agtgtcacct
     3601 aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3661 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3721 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3781 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3841 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3901 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3961 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4021 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4081 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4141 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4201 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4261 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4321 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4381 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4441 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4501 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4561 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4621 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4681 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4741 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4801 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4861 cgtgtagata attacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4921 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4981 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5041 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5101 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5161 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5221 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5281 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5341 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5401 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5461 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5521 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5581 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5641 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5701 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5761 aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     5821 tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5881 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5941 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     6001 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     6061 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     6121 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     6181 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     6241 caccacaccc gccgcactta atgcgccgct acagggcgcg tccattcgcc attcaggctg
     6301 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     6361 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     6421 tgtaaaacga cggccagtga attgtaatac gactcactat a
//
