LOCUS       sequence-374818-        6265 bp    DNA     circular SYN 15-APR-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 6265)
  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 6265)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-spGFP(1-10)
FEATURES             Location/Qualifiers
     source          1..6265
                     /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..2000
                     /codon_start=1
                     /product="fragment of superfolder GFP consisting of the 
                     first 10 beta-strands"
                     /label=GFP(1-10)
                     /note="associates with GFP11, the 11th beta-strand, to 
                     reconstitute fluorescent GFP (Cabantous et al., 2005; 
                     Kamiyama et al., 2016)"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGK
                     YKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK"
     CDS             2403..3197
                     /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(3390..3408)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3391..3408)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3426..3442)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3426..3442)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3439..3461)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3450..3466
                     /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(3474..3480,3481..3498,3499..3504))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3519..3540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3657..3674)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3828..4416)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3908..3927)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4587..5378,5379..5447))
                     /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     5210..5229
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5448..5552)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5630..6085)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5767..5788)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5979..5998
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6211..6233
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6225..6242
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6226..6242
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6249..6265,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6249..6265,1..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 ttccaaagga gaagaactgt
     1381 ttaccggtgt tgtgccaatt ttggttgaac tcgatggtga tgtcaacgga cataagttct
     1441 cagtgagagg cgaaggagaa ggtgacgcca ccattggaaa attgactctt aaattcatct
     1501 gtactactgg taaacttcct gtaccatggc cgactctcgt aacaacgctt acgtacggag
     1561 ttcagtgctt ttcgagatac ccagaccata tgaaaagaca tgactttttt aagtcggcta
     1621 tgcctgaagg ttacgtgcaa gaaagaacaa tttcgttcaa agatgatgga aaatataaaa
     1681 ctagagcagt tgttaaattt gaaggagata ctttggttaa ccgcattgaa ctgaaaggaa
     1741 cagattttaa agaagatggt aatattcttg gacacaaact cgaatacaat tttaatagtc
     1801 ataacgtata catcactgct gataagcaaa agaacggaat taaagcgaat ttcacagtac
     1861 gccataatgt agaagatggc agtgttcaac ttgccgacca ttaccaacaa aacaccccta
     1921 ttggagacgg tccggtactt cttcctgata atcactacct ctcaacacaa acagtcctga
     1981 gcaaagatcc aaatgaaaaa ggatccggat caggatctgg atcaggatcg ggtagttaag
     2041 agctccgctg cgcttaaatg tctttcatta taatcaatgt ataaccttta tgtagtattt
     2101 caatctatgc cgctgtgtac gtgcactgcg gtgcctatcc ttcggcatta gagagtcact
     2161 gtttgtgtag atcgtagctg catgtctgac atctggttca ttagtgccgt tttgagtctg
     2221 cactgttgtg caacttgata ttcatcactg cacaccaacc gtgtgtttcc tcctttgggt
     2281 gcacccttta atccttgtct tctccttttt tgtctctttc cccctcgaaa aggtgtcaaa
     2341 ctactgccgc ataaactacg gttatcccaa atttaagaga aagcaataaa gcatcagcta
     2401 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     2461 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     2521 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     2581 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     2641 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     2701 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     2761 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     2821 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     2881 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     2941 acggcgagga tctcgtcgtg acccacggcg atgcctgctt gccgaatatc atggtggaaa
     3001 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3061 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3121 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3181 ttgacgagtt cttctgactc gagtgcccat ttagttggct tttcccttgt ctcgtgtctt
     3241 ttccgtggaa aggttcccgg agtaatctga tggcacagca gggaggtgcg cctgcaggtt
     3301 ggttaggaag gggggatgat gtaaaagaag aaaatggggg gattcgagcc ccgcacaggt
     3361 ctctcaaatt ggatgcatag cttgagtatt ctatagtgtc acctaaatag cttggcgtaa
     3421 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3481 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3541 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3601 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3661 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3721 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3781 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3841 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3901 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3961 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4021 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4081 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4141 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4201 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4261 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4321 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4381 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4441 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4501 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4561 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4621 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataattacg
     4681 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4741 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4801 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4861 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4921 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4981 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5041 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5101 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5161 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5221 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5281 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5341 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5401 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5461 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5521 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgat
     5581 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     5641 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     5701 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     5761 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     5821 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     5881 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     5941 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     6001 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgca
     6061 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     6121 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     6181 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     6241 gtgaattgta atacgactca ctata
//
