LOCUS       sequence-399923-        6355 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 6355)
  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 6355)
  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-g418-tagRFPt
FEATURES             Location/Qualifiers
     source          1..6355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             171..965
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2493..3287
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(3480..3498)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3481..3498)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3516..3532)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3516..3532)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3529..3551)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3540..3556
                     /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(3564..3570,3571..3588,3589..3594))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3609..3630
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3747..3764)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3918..4506)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3998..4017)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4677..5468,5469..5537))
                     /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     5300..5319
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5538..5642)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5720..6175)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5857..5878)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6069..6088
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6301..6323
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6315..6332
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6316..6332
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6339..6355,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6339..6355,1..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 atgattgaac
      181 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
      241 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
      301 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
      361 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
      421 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
      481 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
      541 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
      601 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
      661 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
      721 tcgtcgtgac ccacggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
      781 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
      841 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
      901 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
      961 tctgaccatg gtgcccattt agttggcctt tcccttgtct cgtgtctttt ccgtggaaag
     1021 gttcccggag taatctgatg gcacagcagg gaggtgcgcc tgcaggttgg ttaggaaggg
     1081 gggatgatgt aaaagaagaa aatgggggga ttcgagcccc acgcgttgct caagctgtgt
     1141 agcgcaggcg tttccttaca tatttctcta acaggcacgg aagcctaaca aatacacttg
     1201 gcttattttt ttgccccctc atgtcttgta caaatatttg cgatagctta gctatcagcc
     1261 acattaatca aacaagtata ccaacaagcc cgaaaacaga aactcaactg caacgggtac
     1321 ctatgggttc tggtagtggt tccggttccg gttctaagct tgtctcgaag ggagaggagc
     1381 ttataaagga aaatatgcac atgaagttat atatggaagg aacggttaat aatcaccatt
     1441 ttaaatgtac gtcagaaggt gaaggaaagc catacgaggg cacacagact atgaggataa
     1501 aggttgtcga gggaggccca ctgcctttcg cgttcgatat tctcgccacg tcttttatgt
     1561 atggctcaag gactttcatc aatcatacgc agggcatccc ggactttttc aagcagagtt
     1621 ttcctgaggg gtttacgtgg gagcgtgtta ccacatacga agatggagga gtgcttaccg
     1681 caactcagga cacttcatta caagacggat gcctgatcta taacgttaag atccgcggag
     1741 tgaatttccc gagtaacgga ccagtgatgc aaaagaaaac acttggatgg gaagctaaca
     1801 cggagatgtt gtatcccgca gatgggggtt tggagggcag gacagacatg gcattaaaat
     1861 tggtgggcgg tggtcacctg atctgtaatt ttaagacgac gtatcgaagt aagaaacctg
     1921 ctaagaactt gaagatgcca ggggtctact acgtcgatca tcgccttgag aggattaaag
     1981 aggcagataa ggagacctat gtagaacagc acgaggtggc tgtggcccgt tactgcgatc
     2041 tgccctcgaa gttaggtcac aaattaaacg gcatggacga attgtacaag ggatccggat
     2101 caggatctgg atcaggatcg ggtagttaag agctccgctg cgcttaaatg tctttcatta
     2161 taatcaatgt ataaccttta tgtagtattt caatctatgc cgctgtgtac gtgcactgcg
     2221 gtgcctatcc ttcggcatta gagagtcact gtttgtgtag atcgtagctg catgtctgac
     2281 atctggttca ttagtgccgt tttgagtctg cactgttgtg caacttgata ttcatcactg
     2341 cacaccaacc gtgtgtttcc tcctttgggt gcacccttta atccttgtct tctccttttt
     2401 tgtctctttc cccctcgaaa aggtgtcaaa ctactgccgc ataaactacg gttatcccaa
     2461 atttaagaga aagcaataaa gcatcagcta gcatgattga acaagatgga ttgcacgcag
     2521 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     2581 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     2641 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     2701 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     2761 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     2821 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     2881 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     2941 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3001 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccacggcg
     3061 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3121 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3181 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3241 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgactc gagtgcccat
     3301 ttagttggct tttcccttgt ctcgtgtctt ttccgtggaa aggttcccgg agtaatctga
     3361 tggcacagca gggaggtgcg cctgcaggtt ggttaggaag gggggatgat gtaaaagaag
     3421 aaaatggggg gattcgagcc ccgcacaggt ctctcaaatt ggatgcatag cttgagtatt
     3481 ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3541 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3601 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3661 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3721 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3781 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3841 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3901 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3961 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4021 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4081 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4141 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4201 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4261 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4321 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4381 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4441 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4501 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4561 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4621 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4681 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4741 gcctgactcc ccgtcgtgta gataattacg atacgggagg gcttaccatc tggccccagt
     4801 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4861 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4921 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4981 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5041 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5101 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5161 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5221 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5281 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5341 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5401 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5461 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5521 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5581 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5641 cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag
     5701 gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     5761 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     5821 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     5881 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     5941 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     6001 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     6061 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     6121 acgctgcgcg taaccaccac acccgccgca cttaatgcgc cgctacaggg cgcgtccatt
     6181 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     6241 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     6301 cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca ctata
//
