LOCUS       sequence-355696-        6100 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 6100)
  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 6100)
  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-g418-10V5
FEATURES             Location/Qualifiers
     source          1..6100
                     /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..1403
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1410..1451
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1458..1499
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1506..1547
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1554..1595
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1602..1643
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1650..1691
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1698..1739
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1746..1787
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1794..1835
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2238..3032
                     /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(3225..3243)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3226..3243)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3261..3277)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3261..3277)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3274..3296)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3285..3301
                     /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(3309..3315,3316..3333,3334..3339))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3354..3375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3492..3509)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3663..4251)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3743..3762)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4422..5213,5214..5282))
                     /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     5045..5064
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5283..5387)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5465..5920)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5602..5623)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5814..5833
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6046..6068
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6060..6077
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6061..6077
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6084..6100,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6084..6100,1..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 tggaaagcca attccaaacc
     1381 ctcttcttgg actcgactcc accggctctg gtaaaccgat cccgaatcca ctccttggat
     1441 tggattctac cggatcgggg aaaccgatac ctaatccgct gctgggcctt gactcgacgg
     1501 gctcgggaaa accaatacct aatccattgc ttggactcga ctcgactggc agtggtaaac
     1561 ccataccgaa tcccttgctg gggctcgact ccacagggtc cgggaagcct atacccaatc
     1621 cgctcctggg tttggattct actggcagtg ggaagcccat tccgaatcct ctgctcggct
     1681 tggacagtac aggctcggga aagcccatcc ccaatccgct gttggggctt gattcaactg
     1741 ggtcgggtaa acccatcccg aatccgcttc tcgggctcga ttcaactggg tctggaaagc
     1801 ccatccctaa cccgcttctc ggtctggaca gcacgggatc cggatcagga tctggatcag
     1861 gatcgggtag ttaagagctc cgctgcgctt aaatgtcttt cattataatc aatgtataac
     1921 ctttatgtag tatttcaatc tatgccgctg tgtacgtgca ctgcggtgcc tatccttcgg
     1981 cattagagag tcactgtttg tgtagatcgt agctgcatgt ctgacatctg gttcattagt
     2041 gccgttttga gtctgcactg ttgtgcaact tgatattcat cactgcacac caaccgtgtg
     2101 tttcctcctt tgggtgcacc ctttaatcct tgtcttctcc ttttttgtct ctttccccct
     2161 cgaaaaggtg tcaaactact gccgcataaa ctacggttat cccaaattta agagaaagca
     2221 ataaagcatc agctagcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     2281 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     2341 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     2401 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     2461 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     2521 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     2581 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     2641 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     2701 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     2761 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca cggcgatgcc tgcttgccga
     2821 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     2881 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     2941 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     3001 ccttctatcg ccttcttgac gagttcttct gactcgagtg cccatttagt tggcttttcc
     3061 cttgtctcgt gtcttttccg tggaaaggtt cccggagtaa tctgatggca cagcagggag
     3121 gtgcgcctgc aggttggtta ggaagggggg atgatgtaaa agaagaaaat ggggggattc
     3181 gagccccgca caggtctctc aaattggatg catagcttga gtattctata gtgtcaccta
     3241 aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3301 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3361 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3421 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3481 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3541 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3601 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3661 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3721 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3781 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3841 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3901 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3961 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4021 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4081 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4141 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4201 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4261 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4321 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4381 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4441 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4501 gtgtagataa ttacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4561 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4621 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4681 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4741 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4801 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4861 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4921 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4981 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5041 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5101 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5161 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5221 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5281 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5341 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5401 aaagtgccac ctgatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     5461 caggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5521 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5581 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5641 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5701 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5761 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5821 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5881 accacacccg ccgcacttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc
     5941 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     6001 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     6061 gtaaaacgac ggccagtgaa ttgtaatacg actcactata
//
