LOCUS       sequence-374841-        6316 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 6316)
  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 6316)
  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-BFP
FEATURES             Location/Qualifiers
     source          1..6316
                     /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             2454..3248
                     /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(3441..3459)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3442..3459)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3477..3493)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3477..3493)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3490..3512)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3501..3517
                     /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(3525..3531,3532..3549,3550..3555))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3570..3591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3708..3725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3879..4467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3959..3978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4638..5429,5430..5498))
                     /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     5261..5280
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5499..5603)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5681..6136)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5818..5839)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6030..6049
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6262..6284
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6276..6293
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6277..6293
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6300..6316,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6300..6316,1..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 tagtgagttg attaaggaaa
     1381 acatgcacat gaagttgtac atggaaggaa cagtagataa tcatcatttc aaatgtacgt
     1441 cagaaggtga gggcaaacct tatgaaggaa ctcaaactat gagaattaaa gtcgttgagg
     1501 gtggcccact accatttgca ttcgatatcc tcgcaacatc ttttctgtat ggttctaaaa
     1561 cctttatcaa ccatacacaa ggcataccag atttctttaa gcaatcattt cctgaaggtt
     1621 tcacatggga aagagtaact acctatgaag atggtggtgt ccttacagct actcaagaca
     1681 cgtctttgca ggatggttgt ttaatctaca atgttaagat aagaggagtg aactttacat
     1741 ccaatggacc agtcatgcaa aagaaaactc taggttggga ggcattcaca gaaactttgt
     1801 acccagccga tgggggctta gaagggagaa atgacatggc tttaaaactg gttggcggta
     1861 gccatttgat tgcgaacgct aaaacaacct acagaagcaa aaagccagct aaaaatctta
     1921 agatgcctgt atactacgtg gattacagat tagaaaggaa ggaagctaat aatgaaactt
     1981 acgttgaaca gcacgaggtg gccgttgccc gttattgcga ccttccttca aaattgggac
     2041 acaaactaaa cggatccgga tcaggatctg gatcaggatc gggtagttaa gagctccgct
     2101 gcgcttaaat gtctttcatt ataatcaatg tataaccttt atgtagtatt tcaatctatg
     2161 ccgctgtgta cgtgcactgc ggtgcctatc cttcggcatt agagagtcac tgtttgtgta
     2221 gatcgtagct gcatgtctga catctggttc attagtgccg ttttgagtct gcactgttgt
     2281 gcaacttgat attcatcact gcacaccaac cgtgtgtttc ctcctttggg tgcacccttt
     2341 aatccttgtc ttctcctttt ttgtctcttt ccccctcgaa aaggtgtcaa actactgccg
     2401 cataaactac ggttatccca aatttaagag aaagcaataa agcatcagct agcatgattg
     2461 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     2521 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     2581 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     2641 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     2701 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     2761 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     2821 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     2881 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     2941 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     3001 atctcgtcgt gacccacggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     3061 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     3121 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3181 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     3241 tcttctgact cgagtgccca tttagttggc ttttcccttg tctcgtgtct tttccgtgga
     3301 aaggttcccg gagtaatctg atggcacagc agggaggtgc gcctgcaggt tggttaggaa
     3361 ggggggatga tgtaaaagaa gaaaatgggg ggattcgagc cccgcacagg tctctcaaat
     3421 tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta atcatggtca
     3481 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3541 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3601 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3661 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3721 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3781 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3841 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3901 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3961 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4021 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4081 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4141 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4201 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4261 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4321 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4381 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4441 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4501 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4561 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4621 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4681 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataattac gatacgggag
     4741 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4801 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4861 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4921 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4981 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5041 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5101 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5161 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5221 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5281 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5341 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5401 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5461 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5521 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5581 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga tgcggtgtga
     5641 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt
     5701 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     5761 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     5821 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     5881 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     5941 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     6001 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     6061 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc acttaatgcg
     6121 ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     6181 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     6241 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt
     6301 aatacgactc actata
//
