LOCUS       sequence-355461-        6319 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 6319)
  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 6319)
  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-mScarlet
FEATURES             Location/Qualifiers
     source          1..6319
                     /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             2457..3251
                     /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(3444..3462)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3445..3462)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3480..3496)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3480..3496)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3493..3515)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3504..3520
                     /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(3528..3534,3535..3552,3553..3558))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3573..3594
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3711..3728)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3882..4470)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3962..3981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4641..5432,5433..5501))
                     /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     5264..5283
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5502..5606)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5684..6139)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5821..5842)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6033..6052
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6265..6287
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6279..6296
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6280..6296
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6303..6319,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6303..6319,1..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 tgtaagcaaa ggggaggctg
     1381 tcataaagga atttatgcga tttaaagtgc acatggaagg gagtatgaac ggccatgagt
     1441 ttgaaataga aggagaggga gaggggcgcc cttacgaagg tactcagacg gcgaagctca
     1501 aagtgactaa gggaggtcct ctgccgttta gctgggatat actttcaccc cagttcatgt
     1561 atggcagccg cgcgttcacg aagcaccccg cagatatccc cgattactat aaacagtcat
     1621 ttcctgaggg gttcaagtgg gaacgagtaa tgaactttga ggatggtggc gctgtaacgg
     1681 taacacaaga tacctcgctc gaagacggta cgctcattta caaagtgaag ctgcgcggaa
     1741 caaatttccc tcctgatgga ccagtaatgc agaaaaagac tatgggatgg gaagcctcga
     1801 cggaacgtct ctacccggag gacggggtgc tgaaagggga tattaaaatg gcactccgac
     1861 tgaaagacgg cggcaggtat cttgctgatt ttaaaaccac gtacaaagct aaaaagccag
     1921 tgcagatgcc tggagcgtat aacgtagacc gaaaactcga cattacgtcg cataacgagg
     1981 actatacggt agtcgaacag tatgagaggt ctgaagggag gcattcaact ggtggtatgg
     2041 atgagttgta taagggatcc ggatcaggat ctggatcagg atcgggtagt taagagctcc
     2101 gctgcgctta aatgtctttc attataatca atgtataacc tttatgtagt atttcaatct
     2161 atgccgctgt gtacgtgcac tgcggtgcct atccttcggc attagagagt cactgtttgt
     2221 gtagatcgta gctgcatgtc tgacatctgg ttcattagtg ccgttttgag tctgcactgt
     2281 tgtgcaactt gatattcatc actgcacacc aaccgtgtgt ttcctccttt gggtgcaccc
     2341 tttaatcctt gtcttctcct tttttgtctc tttccccctc gaaaaggtgt caaactactg
     2401 ccgcataaac tacggttatc ccaaatttaa gagaaagcaa taaagcatca gctagcatga
     2461 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     2521 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     2581 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     2641 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     2701 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     2761 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     2821 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     2881 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     2941 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     3001 aggatctcgt cgtgacccac ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3061 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3121 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3181 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3241 agttcttctg actcgagtgc ccatttagtt ggcttttccc ttgtctcgtg tcttttccgt
     3301 ggaaaggttc ccggagtaat ctgatggcac agcagggagg tgcgcctgca ggttggttag
     3361 gaagggggga tgatgtaaaa gaagaaaatg gggggattcg agccccgcac aggtctctca
     3421 aattggatgc atagcttgag tattctatag tgtcacctaa atagcttggc gtaatcatgg
     3481 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     3541 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     3601 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3661 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3721 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3781 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3841 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3901 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3961 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4021 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4081 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4141 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4201 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4261 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4321 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4381 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4441 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4501 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4561 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4621 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4681 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataat tacgatacgg
     4741 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4801 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4861 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4921 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4981 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5041 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5101 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5161 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5221 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5281 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5341 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5401 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5461 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5521 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5581 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgatgcggtg
     5641 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat
     5701 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     5761 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5821 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5881 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5941 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     6001 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     6061 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcacttaat
     6121 gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     6181 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     6241 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat
     6301 tgtaatacga ctcactata
//
