LOCUS       sequence-399939-        7021 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 7021)
  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 7021)
  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-hygro-3Ty:tagRFPt:3Ty
FEATURES             Location/Qualifiers
     source          1..7021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             171..1196
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             171..1196
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2928..3953
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2928..3953
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(4146..4164)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4147..4164)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4182..4198)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4182..4198)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4195..4217)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4206..4222
                     /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(4230..4236,4237..4254,4255..4260))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4275..4296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4413..4430)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4584..5172)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4664..4683)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5343..6134,6135..6203))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5966..5985
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6204..6308)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6386..6841)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6523..6544)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6735..6754
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6967..6989
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6981..6998
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6982..6998
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(7005..7021,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(7005..7021,1..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 atgaaaaagc
      181 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
      241 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
      301 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
      361 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattta
      421 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
      481 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggcaatggat gcgatcgctg
      541 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
      601 acactacatg gcgtgatttt atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
      661 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
      721 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
      781 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
      841 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
      901 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgcct cggctccggg
      961 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     1021 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     1081 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     1141 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagccat
     1201 ggtgcccatt tagttggctt ttcccttgtc tcgtgtcttt tccgtggaaa ggttcccgga
     1261 gtaatctgat ggcacagcag ggaggtgcgc ctgcaggttg gttaggaagg ggggatgatg
     1321 taaaagaaga aaatgggggg attcgagccc cacgcgttgc tcaagctgtg tagcgcaggc
     1381 gtttccttac atatttctct aacaggcacg gaagcctaac aaatacactt ggcttatttt
     1441 tttgccccct catgtcttgt acaaatattt gcgatagctt agctatcagc cacattaatc
     1501 aaacaagtat accaacaagc ccgaaaacag aaactcaact gcaacgggta cctatgggtt
     1561 ctggtagtgg ttccggttcc ggttctgagg tacacacaaa ccaggaccca ctggacggct
     1621 ccgaggtgca taccaatcaa gatccgctgg acggctctga agttcacacc aatcaagatc
     1681 ccttggataa gcttgtctcg aagggagagg agcttataaa ggaaaatatg cacatgaagt
     1741 tatatatgga aggaacggtt aataatcacc attttaaatg tacgtcagaa ggtgaaggaa
     1801 agccatacga gggcacacag actatgagga taaaggttgt cgagggaggc ccactgcctt
     1861 tcgcgttcga tattctcgcc acgtctttta tgtatggctc aaggactttc atcaatcata
     1921 cgcagggcat cccggacttt ttcaagcaga gttttcctga ggggtttacg tgggagcgtg
     1981 ttaccacata cgaagatgga ggagtgctta ccgcaactca ggacacttca ttacaagacg
     2041 gatgcctgat ctataacgtt aagatccgcg gagtgaattt cccgagtaac ggaccagtga
     2101 tgcaaaagaa aacacttgga tgggaagcta acacggagat gttgtatccc gcagatgggg
     2161 gtttggaggg caggacagac atggcattaa aattggtggg cggtggtcac ctgatctgta
     2221 attttaagac gacgtatcga agtaagaaac ctgctaagaa cttgaagatg ccaggggtct
     2281 actacgtcga tcatcgcctt gagaggatta aagaggcaga taaggagacc tatgtagaac
     2341 agcacgaggt ggctgtggcc cgttactgcg atctgccctc gaagttaggt cacaaattaa
     2401 acggcatgga cgaattgtac aagggatccg aggtacatac aaatcaggac cccctcgatg
     2461 gtagtgaagt ccatacaaat caggacccgc tggatggctc agaggttcat acgaaccaag
     2521 atcctcttga tggatcagga tctggatcag gatcgggtag ttaagagctc cgctgcgctt
     2581 aaatgtcttt cattataatc aatgtataac ctttatgtag tatttcaatc tatgccgctg
     2641 tgtacgtgca ctgcggtgcc tatccttcgg cattagagag tcactgtttg tgtagatcgt
     2701 agctgcatgt ctgacatctg gttcattagt gccgttttga gtctgcactg ttgtgcaact
     2761 tgatattcat cactgcacac caaccgtgtg tttcctcctt tgggtgcacc ctttaatcct
     2821 tgtcttctcc ttttttgtct ctttccccct cgaaaaggtg tcaaactact gccgcataaa
     2881 ctacggttat cccaaattta agagaaagca ataaagcatc agctagcatg aaaaagcctg
     2941 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
     3001 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
     3061 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
     3121 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaatttagcg
     3181 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
     3241 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc aatggatgcg atcgctgcgg
     3301 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
     3361 ctacatggcg tgattttata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
     3421 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
     3481 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
     3541 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     3601 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     3661 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgcctcgg ctccgggcgt
     3721 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     3781 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     3841 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
     3901 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagctcgagt
     3961 gcccatttag ttggcttttc ccttgtctcg tgtcttttcc gtggaaaggt tcccggagta
     4021 atctgatggc acagcaggga ggtgcgcctg caggttggtt aggaaggggg gatgatgtaa
     4081 aagaagaaaa tggggggatt cgagccccgc acaggtctct caaattggat gcatagcttg
     4141 agtattctat agtgtcacct aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4201 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4261 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4321 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4381 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4441 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4501 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4561 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4621 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4681 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4741 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4801 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4861 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4921 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4981 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5041 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5101 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5161 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5221 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5281 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5341 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5401 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5461 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5521 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5581 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5641 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5701 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5761 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5821 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5881 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5941 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6001 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6061 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6121 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6181 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6241 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6301 gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg
     6361 cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg
     6421 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     6481 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     6541 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     6601 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     6661 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     6721 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     6781 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     6841 tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     6901 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     6961 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat
     7021 a
//
