LOCUS       sequence-355332-        5950 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 5950)
  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 5950)
  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-g418-10HA
FEATURES             Location/Qualifiers
     source          1..5950
                     /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             1362..1388
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1395..1421
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1428..1454
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1461..1487
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1494..1520
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1527..1553
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1560..1586
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1593..1619
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1626..1652
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1659..1685
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2088..2882
                     /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(3075..3093)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3076..3093)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3111..3127)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3111..3127)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3124..3146)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3135..3151
                     /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(3159..3165,3166..3183,3184..3189))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3204..3225
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3342..3359)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3513..4101)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3593..3612)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4272..5063,5064..5132))
                     /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     4895..4914
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5133..5237)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5315..5770)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5452..5473)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5664..5683
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5896..5918
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5910..5927
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5911..5927
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5934..5950,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5934..5950,1..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 ttatccttat gatgtgccag
     1381 attacgctgg ttcctatccc tatgacgttc ctgactacgc tgggtcgtac ccttatgatg
     1441 tgccggatta cgccggaagc tacccatacg atgtaccgga ctacgcaggg tcatatcctt
     1501 acgatgttcc agactatgct ggctcctatc cgtacgatgt ccccgactat gcaggatcat
     1561 acccgtatga cgttccagat tatgccgggt cgtaccctta tgacgtccca gattatgccg
     1621 gttcttatcc gtatgatgtg ccggactacg cggggtccta tccgtatgat gtacccgatt
     1681 acgcgggatc cggatcagga tctggatcag gatcgggtag ttaagagctc cgctgcgctt
     1741 aaatgtcttt cattataatc aatgtataac ctttatgtag tatttcaatc tatgccgctg
     1801 tgtacgtgca ctgcggtgcc tatccttcgg cattagagag tcactgtttg tgtagatcgt
     1861 agctgcatgt ctgacatctg gttcattagt gccgttttga gtctgcactg ttgtgcaact
     1921 tgatattcat cactgcacac caaccgtgtg tttcctcctt tgggtgcacc ctttaatcct
     1981 tgtcttctcc ttttttgtct ctttccccct cgaaaaggtg tcaaactact gccgcataaa
     2041 ctacggttat cccaaattta agagaaagca ataaagcatc agctagcatg attgaacaag
     2101 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     2161 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     2221 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     2281 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     2341 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     2401 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     2461 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     2521 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     2581 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     2641 tcgtgaccca cggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     2701 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     2761 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     2821 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     2881 gactcgagtg cccatttagt tggcttttcc cttgtctcgt gtcttttccg tggaaaggtt
     2941 cccggagtaa tctgatggca cagcagggag gtgcgcctgc aggttggtta ggaagggggg
     3001 atgatgtaaa agaagaaaat ggggggattc gagccccgca caggtctctc aaattggatg
     3061 catagcttga gtattctata gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg
     3121 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     3181 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     3241 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3301 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3361 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3421 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3481 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3541 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3601 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3661 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3721 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3781 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3841 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3901 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3961 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4021 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4081 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4141 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4201 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4261 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4321 cgttcatcca tagttgcctg actccccgtc gtgtagataa ttacgatacg ggagggctta
     4381 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4441 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4501 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4561 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4621 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4681 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4741 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4801 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4861 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4921 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4981 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5041 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5101 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5161 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5221 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgatgcggt gtgaaatacc
     5281 gcacagatgc gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta
     5341 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     5401 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     5461 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     5521 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     5581 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     5641 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     5701 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcacttaa tgcgccgcta
     5761 cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     5821 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     5881 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg
     5941 actcactata
//
