LOCUS       sequence-355465-        6322 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 6322)
  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 6322)
  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-mScarletI
FEATURES             Location/Qualifiers
     source          1..6322
                     /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             2460..3254
                     /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(3447..3465)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3448..3465)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3483..3499)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3483..3499)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3496..3518)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3507..3523
                     /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(3531..3537,3538..3555,3556..3561))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3576..3597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3714..3731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3885..4473)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3965..3984)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4644..5435,5436..5504))
                     /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     5267..5286
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5505..5609)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5687..6142)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5824..5845)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6036..6055
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6268..6290
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6282..6299
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6283..6299
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6306..6322,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6306..6322,1..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 tatggtatca aagggcgaag
     1381 cagtaatcaa ggagttcatg cggtttaagg tacacatgga gggttccatg aatggccatg
     1441 aatttgagat cgaaggagag ggggaagggc gtccttacga aggaacacaa acagctaaac
     1501 ttaaggtaac taagggggga cccctcccct tttcctggga catactgtca ccacaattca
     1561 tgtacggcag ccgagccttt ataaaacatc cagctgatat acccgattat tataagcaaa
     1621 gttttcccga gggctttaag tgggaaaggg tcatgaactt cgaggacggg ggtgcggtaa
     1681 ctgtaaccca agacacctct ctcgaagacg ggacgcttat ttataaggtg aaactccggg
     1741 ggaccaattt tccccctgac ggacctgtca tgcagaagaa gacaatgggc tgggaggcgt
     1801 ccacagagcg cctgtatccg gaggatggcg tacttaaggg tgacataaaa atggcacttc
     1861 gtttgaagga cggtggccgt tatctcgctg actttaagac aacttataag gcgaaaaagc
     1921 cggtacagat gccaggagca tacaatgttg accgcaaatt ggacatcacc agccacaacg
     1981 aagactatac cgtcgttgaa cagtacgaac ggagtgaagg gaggcacagt acaggcggga
     2041 tggacgaatt gtataaagga tccggatcag gatctggatc aggatcgggt agttaagagc
     2101 tccgctgcgc ttaaatgtct ttcattataa tcaatgtata acctttatgt agtatttcaa
     2161 tctatgccgc tgtgtacgtg cactgcggtg cctatccttc ggcattagag agtcactgtt
     2221 tgtgtagatc gtagctgcat gtctgacatc tggttcatta gtgccgtttt gagtctgcac
     2281 tgttgtgcaa cttgatattc atcactgcac accaaccgtg tgtttcctcc tttgggtgca
     2341 ccctttaatc cttgtcttct ccttttttgt ctctttcccc ctcgaaaagg tgtcaaacta
     2401 ctgccgcata aactacggtt atcccaaatt taagagaaag caataaagca tcagctagca
     2461 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     2521 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     2581 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     2641 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     2701 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     2761 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     2821 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     2881 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     2941 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     3001 gcgaggatct cgtcgtgacc cacggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     3061 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     3121 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     3181 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     3241 acgagttctt ctgactcgag tgcccattta gttggctttt cccttgtctc gtgtcttttc
     3301 cgtggaaagg ttcccggagt aatctgatgg cacagcaggg aggtgcgcct gcaggttggt
     3361 taggaagggg ggatgatgta aaagaagaaa atggggggat tcgagccccg cacaggtctc
     3421 tcaaattgga tgcatagctt gagtattcta tagtgtcacc taaatagctt ggcgtaatca
     3481 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     3541 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3601 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3661 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3721 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3781 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3841 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3901 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3961 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4021 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4081 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4141 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4201 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4261 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4321 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4381 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4441 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4501 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4561 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4621 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4681 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aattacgata
     4741 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4801 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4861 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4921 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4981 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5041 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5101 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5161 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5221 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5281 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5341 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5401 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5461 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5521 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5581 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg
     5641 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt
     5701 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     5761 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     5821 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     5881 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     5941 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     6001 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     6061 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcactt
     6121 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc
     6181 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     6241 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     6301 aattgtaata cgactcacta ta
//
