LOCUS       sequence-400015-        6526 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 6526)
  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 6526)
  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:mScarletI:3Ty
FEATURES             Location/Qualifiers
     source          1..6526
                     /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             2664..3458
                     /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(3651..3669)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3652..3669)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3687..3703)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3687..3703)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3700..3722)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3711..3727
                     /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(3735..3741,3742..3759,3760..3765))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3780..3801
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3918..3935)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4089..4677)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4169..4188)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4848..5639,5640..5708))
                     /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     5471..5490
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5709..5813)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5891..6346)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6028..6049)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6240..6259
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6472..6494
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6486..6503
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6487..6503
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6510..6526,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6510..6526,1..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 cttatggtat caaagggcga agcagtaatc aaggagttca
     1501 tgcggtttaa ggtacacatg gagggttcca tgaatggcca tgaatttgag atcgaaggag
     1561 agggggaagg gcgtccttac gaaggaacac aaacagctaa acttaaggta actaaggggg
     1621 gacccctccc cttttcctgg gacatactgt caccacaatt catgtacggc agccgagcct
     1681 ttataaaaca tccagctgat atacccgatt attataagca aagttttccc gagggcttta
     1741 agtgggaaag ggtcatgaac ttcgaggacg ggggtgcggt aactgtaacc caagacacct
     1801 ctctcgaaga cgggacgctt atttataagg tgaaactccg ggggaccaat tttccccctg
     1861 acggacctgt catgcagaag aagacaatgg gctgggaggc gtccacagag cgcctgtatc
     1921 cggaggatgg cgtacttaag ggtgacataa aaatggcact tcgtttgaag gacggtggcc
     1981 gttatctcgc tgactttaag acaacttata aggcgaaaaa gccggtacag atgccaggag
     2041 catacaatgt tgaccgcaaa ttggacatca ccagccacaa cgaagactat accgtcgttg
     2101 aacagtacga acggagtgaa gggaggcaca gtacaggcgg gatggacgaa ttgtataaag
     2161 gatccgaggt acatacaaat caggaccccc tcgatggtag tgaagtccat acaaatcagg
     2221 acccgctgga tggctcagag gttcatacga accaagatcc tcttgatgga tcaggatctg
     2281 gatcaggatc gggtagttaa gagctccgct gcgcttaaat gtctttcatt ataatcaatg
     2341 tataaccttt atgtagtatt tcaatctatg ccgctgtgta cgtgcactgc ggtgcctatc
     2401 cttcggcatt agagagtcac tgtttgtgta gatcgtagct gcatgtctga catctggttc
     2461 attagtgccg ttttgagtct gcactgttgt gcaacttgat attcatcact gcacaccaac
     2521 cgtgtgtttc ctcctttggg tgcacccttt aatccttgtc ttctcctttt ttgtctcttt
     2581 ccccctcgaa aaggtgtcaa actactgccg cataaactac ggttatccca aatttaagag
     2641 aaagcaataa agcatcagct agcatgattg aacaagatgg attgcacgca ggttctccgg
     2701 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     2761 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     2821 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     2881 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     2941 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3001 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3061 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3121 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3181 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccacggc gatgcctgct
     3241 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3301 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3361 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3421 gcatcgcctt ctatcgcctt cttgacgagt tcttctgact cgagtgccca tttagttggc
     3481 ttttcccttg tctcgtgtct tttccgtgga aaggttcccg gagtaatctg atggcacagc
     3541 agggaggtgc gcctgcaggt tggttaggaa ggggggatga tgtaaaagaa gaaaatgggg
     3601 ggattcgagc cccgcacagg tctctcaaat tggatgcata gcttgagtat tctatagtgt
     3661 cacctaaata gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3721 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3781 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3841 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3901 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3961 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4021 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4081 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4141 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4201 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4261 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4321 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4381 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4441 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4501 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4561 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4621 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4681 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4741 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4801 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4861 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4921 cccgtcgtgt agataattac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4981 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5041 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5101 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5161 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5221 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5281 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5341 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5401 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5461 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5521 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5581 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5641 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5701 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5761 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5821 ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     5881 ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     5941 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     6001 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     6061 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     6121 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     6181 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     6241 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     6301 gtaaccacca cacccgccgc acttaatgcg ccgctacagg gcgcgtccat tcgccattca
     6361 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     6421 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     6481 gacgttgtaa aacgacggcc agtgaattgt aatacgactc actata
//
