LOCUS       sequence-374840-        5677 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 5677)
  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 5677)
  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-spGFP(11)
FEATURES             Location/Qualifiers
     source          1..5677
                     /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             1365..1412
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             1815..2609
                     /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(2802..2820)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2803..2820)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2838..2854)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2838..2854)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2851..2873)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2862..2878
                     /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(2886..2892,2893..2910,2911..2916))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2931..2952
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3069..3086)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3240..3828)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3320..3339)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3999..4790,4791..4859))
                     /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     4622..4641
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4860..4964)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5042..5497)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5179..5200)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5391..5410
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5623..5645
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5637..5654
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5638..5654
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5661..5677,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5661..5677,1..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 tatgcgtgac cacatggtcc
     1381 ttcatgagta tgtaaatgct gccggtatca ccggatccgg atcaggatct ggatcaggat
     1441 cgggtagtta agagctccgc tgcgcttaaa tgtctttcat tataatcaat gtataacctt
     1501 tatgtagtat ttcaatctat gccgctgtgt acgtgcactg cggtgcctat ccttcggcat
     1561 tagagagtca ctgtttgtgt agatcgtagc tgcatgtctg acatctggtt cattagtgcc
     1621 gttttgagtc tgcactgttg tgcaacttga tattcatcac tgcacaccaa ccgtgtgttt
     1681 cctcctttgg gtgcaccctt taatccttgt cttctccttt tttgtctctt tccccctcga
     1741 aaaggtgtca aactactgcc gcataaacta cggttatccc aaatttaaga gaaagcaata
     1801 aagcatcagc tagcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     1861 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     1921 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     1981 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     2041 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     2101 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     2161 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     2221 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     2281 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     2341 cgcgcatgcc cgacggcgag gatctcgtcg tgacccacgg cgatgcctgc ttgccgaata
     2401 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     2461 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     2521 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     2581 tctatcgcct tcttgacgag ttcttctgac tcgagtgccc atttagttgg cttttccctt
     2641 gtctcgtgtc ttttccgtgg aaaggttccc ggagtaatct gatggcacag cagggaggtg
     2701 cgcctgcagg ttggttagga aggggggatg atgtaaaaga agaaaatggg gggattcgag
     2761 ccccgcacag gtctctcaaa ttggatgcat agcttgagta ttctatagtg tcacctaaat
     2821 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2881 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2941 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3001 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3061 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3121 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3181 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3241 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3301 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3361 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3421 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3481 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3541 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3601 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3661 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3721 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3781 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3841 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3901 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3961 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4021 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4081 tagataatta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4141 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4201 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4261 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4321 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4381 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4441 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4501 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4561 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4621 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4681 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4741 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4801 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4861 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4921 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4981 gtgccacctg atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     5041 gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     5101 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     5161 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     5221 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     5281 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     5341 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     5401 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     5461 acacccgccg cacttaatgc gccgctacag ggcgcgtcca ttcgccattc aggctgcgca
     5521 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     5581 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     5641 aaacgacggc cagtgaattg taatacgact cactata
//
