LOCUS       sequence-374794-        5461 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 5461)
  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 5461)
  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: pPOTv7-blast-spGFP(1-10)
FEATURES             Location/Qualifiers
     source          1..5461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             960..1598
                     /codon_start=1
                     /product="fragment of superfolder GFP consisting of the 
                     first 10 beta-strands"
                     /label=GFP(1-10)
                     /note="associates with GFP11, the 11th beta-strand, to 
                     reconstitute fluorescent GFP (Cabantous et al., 2005; 
                     Kamiyama et al., 2016)"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGK
                     YKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK"
     promoter        complement(2586..2604)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2587..2604)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2622..2638)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2622..2638)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2635..2657)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2646..2662
                     /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(2670..2676,2677..2694,2695..2700))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2715..2736
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2853..2870)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3024..3612)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3104..3123)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3783..4574,4575..4643))
                     /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     4406..4425
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4644..4748)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4826..5281)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4963..4984)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5175..5194
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5407..5429
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5421..5438
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5422..5438
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5445..5461,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5445..5461,1..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 tctaagcttt
      961 ccaaaggaga agaactgttt accggtgttg tgccaatttt ggttgaactc gatggtgatg
     1021 tcaacggaca taagttctca gtgagaggcg aaggagaagg tgacgccacc attggaaaat
     1081 tgactcttaa attcatctgt actactggta aacttcctgt accatggccg actctcgtaa
     1141 caacgcttac gtacggagtt cagtgctttt cgagataccc agaccatatg aaaagacatg
     1201 acttttttaa gtcggctatg cctgaaggtt acgtgcaaga aagaacaatt tcgttcaaag
     1261 atgatggaaa atataaaact agagcagttg ttaaatttga aggagatact ttggttaacc
     1321 gcattgaact gaaaggaaca gattttaaag aagatggtaa tattcttgga cacaaactcg
     1381 aatacaattt taatagtcat aacgtataca tcactgctga taagcaaaag aacggaatta
     1441 aagcgaattt cacagtacgc cataatgtag aagatggcag tgttcaactt gccgaccatt
     1501 accaacaaaa cacccctatt ggagacggtc cggtacttct tcctgataat cactacctct
     1561 caacacaaac agtcctgagc aaagatccaa atgaaaaagg atccggatca ggatctggat
     1621 caggatcggg tagttaagag ctccgctgcg cttaaatgtc tttcattata atcaatgtat
     1681 aacctttatg tagtatttca atctatgccg ctgtgtacgt gcactgcggt gcctatcctt
     1741 cggcattaga gagtcactgt ttgtgtagat cgtagctgca tgtctgacat ctggttcatt
     1801 agtgccgttt tgagtctgca ctgttgtgca acttgatatt catcactgca caccaaccgt
     1861 gtgtttcctc ctttgggtgc accctttaat ccttgtcttc tccttttttg tctctttccc
     1921 cctcgaaaag gtgtcaaact actgccgcat aaactacggt tatcccaaat ttaagagaaa
     1981 gcaataaagc atcagctagc atgcctttgt ctcaagaaga atccaccctc attgaaagag
     2041 caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc
     2101 tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt
     2161 gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta
     2221 tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg
     2281 tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga
     2341 cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taactcgagt
     2401 gcccatttag ttggcttttc ccttgtctcg tgtcttttcc gtggaaaggt tcccggagta
     2461 atctgatggc acagcaggga ggtgcgcctg caggttggtt aggaaggggg gatgatgtaa
     2521 aagaagaaaa tggggggatt cgagccccgc acaggtctct caaattggat gcatagcttg
     2581 agtattctat agtgtcacct aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg
     2641 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     2701 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     2761 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     2821 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     2881 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     2941 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3001 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3061 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3121 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3181 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3241 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3301 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3361 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3421 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3481 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3541 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3601 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3661 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     3721 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     3781 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     3841 atagttgcct gactccccgt cgtgtagata attacgatac gggagggctt accatctggc
     3901 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     3961 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4021 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4081 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4141 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4201 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     4261 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4321 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     4381 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     4441 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     4501 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     4561 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     4621 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     4681 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     4741 gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg
     4801 cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg
     4861 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     4921 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     4981 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     5041 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     5101 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     5161 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     5221 gcggtcacgc tgcgcgtaac caccacaccc gccgcactta atgcgccgct acagggcgcg
     5281 tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     5341 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     5401 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat
     5461 a
//
