LOCUS       sequence-400076-        6358 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 6358)
  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 6358)
  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-His:mScarletI:His
FEATURES             Location/Qualifiers
     source          1..6358
                     /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             1356..1373
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2082..2099
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2496..3290
                     /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(3483..3501)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3484..3501)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3519..3535)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3519..3535)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3532..3554)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3543..3559
                     /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(3567..3573,3574..3591,3592..3597))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3612..3633
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3750..3767)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3921..4509)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4001..4020)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4680..5471,5472..5540))
                     /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     5303..5322
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5541..5645)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5723..6178)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5860..5881)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6072..6091
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6304..6326
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6318..6335
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6319..6335
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6342..6358,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6342..6358,1..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 gttctcacca ccaccaccat cacaagctta
     1381 tggtatcaaa gggcgaagca gtaatcaagg agttcatgcg gtttaaggta cacatggagg
     1441 gttccatgaa tggccatgaa tttgagatcg aaggagaggg ggaagggcgt ccttacgaag
     1501 gaacacaaac agctaaactt aaggtaacta aggggggacc cctccccttt tcctgggaca
     1561 tactgtcacc acaattcatg tacggcagcc gagcctttat aaaacatcca gctgatatac
     1621 ccgattatta taagcaaagt tttcccgagg gctttaagtg ggaaagggtc atgaacttcg
     1681 aggacggggg tgcggtaact gtaacccaag acacctctct cgaagacggg acgcttattt
     1741 ataaggtgaa actccggggg accaattttc cccctgacgg acctgtcatg cagaagaaga
     1801 caatgggctg ggaggcgtcc acagagcgcc tgtatccgga ggatggcgta cttaagggtg
     1861 acataaaaat ggcacttcgt ttgaaggacg gtggccgtta tctcgctgac tttaagacaa
     1921 cttataaggc gaaaaagccg gtacagatgc caggagcata caatgttgac cgcaaattgg
     1981 acatcaccag ccacaacgaa gactataccg tcgttgaaca gtacgaacgg agtgaaggga
     2041 ggcacagtac aggcgggatg gacgaattgt ataaaggatc ccaccaccac caccatcacg
     2101 gatcaggatc tggatcagga tcgggtagtt aagagctccg ctgcgcttaa atgtctttca
     2161 ttataatcaa tgtataacct ttatgtagta tttcaatcta tgccgctgtg tacgtgcact
     2221 gcggtgccta tccttcggca ttagagagtc actgtttgtg tagatcgtag ctgcatgtct
     2281 gacatctggt tcattagtgc cgttttgagt ctgcactgtt gtgcaacttg atattcatca
     2341 ctgcacacca accgtgtgtt tcctcctttg ggtgcaccct ttaatccttg tcttctcctt
     2401 ttttgtctct ttccccctcg aaaaggtgtc aaactactgc cgcataaact acggttatcc
     2461 caaatttaag agaaagcaat aaagcatcag ctagcatgat tgaacaagat ggattgcacg
     2521 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     2581 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     2641 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     2701 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     2761 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     2821 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     2881 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     2941 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     3001 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccacg
     3061 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     3121 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     3181 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     3241 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga ctcgagtgcc
     3301 catttagttg gcttttccct tgtctcgtgt cttttccgtg gaaaggttcc cggagtaatc
     3361 tgatggcaca gcagggaggt gcgcctgcag gttggttagg aaggggggat gatgtaaaag
     3421 aagaaaatgg ggggattcga gccccgcaca ggtctctcaa attggatgca tagcttgagt
     3481 attctatagt gtcacctaaa tagcttggcg taatcatggt catagctgtt tcctgtgtga
     3541 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3601 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3661 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3721 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3781 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3841 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3901 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3961 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4021 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4081 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4141 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4201 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4261 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4321 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4381 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4441 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4501 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4561 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4621 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4681 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4741 gttgcctgac tccccgtcgt gtagataatt acgatacggg agggcttacc atctggcccc
     4801 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4861 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4921 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4981 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5041 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5101 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5161 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5221 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5281 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5341 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5401 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5461 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5521 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5581 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5641 ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt gaaataccgc acagatgcgt
     5701 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta
     5761 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     5821 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     5881 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     5941 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     6001 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     6061 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     6121 gtcacgctgc gcgtaaccac cacacccgcc gcacttaatg cgccgctaca gggcgcgtcc
     6181 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     6241 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     6301 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactata
//
