LOCUS       sequence-355730-        6535 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 6535)
  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 6535)
  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-g418-3Ty:mNG:3Ty
FEATURES             Location/Qualifiers
     source          1..6535
                     /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             2673..3467
                     /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(3660..3678)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3661..3678)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3696..3712)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3696..3712)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3709..3731)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3720..3736
                     /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(3744..3750,3751..3768,3769..3774))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3789..3810
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3927..3944)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4098..4686)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4178..4197)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4857..5648,5649..5717))
                     /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     5480..5499
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5718..5822)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5900..6355)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6037..6058)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6249..6268
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6481..6503
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6495..6512
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6496..6512
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6519..6535,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6519..6535,1..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 gttctgaggt acacacaaac caggacccac
     1381 tggacggctc cgaggtgcat accaatcaag atccgctgga cggctctgaa gttcacacca
     1441 atcaagatcc cttggataag cttgtctcga aaggtgagga agacaatatg gccagcttgc
     1501 cagcaacgca cgagcttcat atatttgggt cgatcaacgg tgtggatttc gacatggtgg
     1561 gtcaaggcac aggtaatcct aatgacgggt atgaagaact caatttgaag agcactaagg
     1621 gagatctcca gttttccccg tggattcttg tgccgcatat tggttatggt tttcatcagt
     1681 acttaccata ccccgatgga atgtcgccct tccaagcagc tatggtggac ggctcggggt
     1741 atcaagtaca tcgcaccatg caatttgagg acggagcatc cctcaccgta aactaccgct
     1801 atacgtatga ggggagccac ataaaaggtg aggctcaagt taaaggcaca ggcttcccag
     1861 ctgacggccc ggtcatgacc aattcattaa ccgctgctga ttggtgccgt tctaagaaga
     1921 cctaccctaa cgacaagacg atcattagta cttttaagtg gagttatacc acaggaaacg
     1981 gaaagcggta tcgaagcact gctcgaacaa cctacacatt tgcgaagcca atggccgcga
     2041 actacttgaa gaaccagcca atgtatgtgt ttcgaaaaac ggaactgaag cattcgaaaa
     2101 ccgagcttaa ttttaaagag tggcagaagg cgtttacaga cgtgatgggg atggacgaat
     2161 tgtataaggg atccgaggta catacaaatc aggaccccct cgatggtagt gaagtccata
     2221 caaatcagga cccgctggat ggctcagagg ttcatacgaa ccaagatcct cttgatggat
     2281 caggatctgg atcaggatcg ggtagttaag agctccgctg cgcttaaatg tctttcatta
     2341 taatcaatgt ataaccttta tgtagtattt caatctatgc cgctgtgtac gtgcactgcg
     2401 gtgcctatcc ttcggcatta gagagtcact gtttgtgtag atcgtagctg catgtctgac
     2461 atctggttca ttagtgccgt tttgagtctg cactgttgtg caacttgata ttcatcactg
     2521 cacaccaacc gtgtgtttcc tcctttgggt gcacccttta atccttgtct tctccttttt
     2581 tgtctctttc cccctcgaaa aggtgtcaaa ctactgccgc ataaactacg gttatcccaa
     2641 atttaagaga aagcaataaa gcatcagcta gcatgattga acaagatgga ttgcacgcag
     2701 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     2761 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     2821 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     2881 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     2941 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3001 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3061 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3121 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3181 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccacggcg
     3241 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3301 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3361 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3421 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgactc gagtgcccat
     3481 ttagttggct tttcccttgt ctcgtgtctt ttccgtggaa aggttcccgg agtaatctga
     3541 tggcacagca gggaggtgcg cctgcaggtt ggttaggaag gggggatgat gtaaaagaag
     3601 aaaatggggg gattcgagcc ccgcacaggt ctctcaaatt ggatgcatag cttgagtatt
     3661 ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3721 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3781 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3841 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3901 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3961 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4021 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4081 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4141 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4201 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4261 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4321 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4381 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4441 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4501 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4561 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4621 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4681 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4741 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4801 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4861 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4921 gcctgactcc ccgtcgtgta gataattacg atacgggagg gcttaccatc tggccccagt
     4981 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5041 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5101 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5161 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5221 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5281 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5341 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5401 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5461 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5521 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5581 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5641 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5701 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5761 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5821 cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag
     5881 gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     5941 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     6001 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     6061 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     6121 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     6181 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     6241 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     6301 acgctgcgcg taaccaccac acccgccgca cttaatgcgc cgctacaggg cgcgtccatt
     6361 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     6421 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     6481 cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca ctata
//
