LOCUS       sequence-495162-        6553 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 6553)
  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 6553)
  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-3HA::FRB::3HA
FEATURES             Location/Qualifiers
     source          1..6553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1009..1027
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1009..1026
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(1220..2245)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(1220..2245)
                     /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             complement(2642..2668)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2675..2701)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2708..2734)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3026..3052)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3059..3085)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3092..3118)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3509..4534)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(3509..4534)
                     /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"
     primer_bind     complement(4702..4721)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4703..4721)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4728..4745)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4728..4744)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4737..4759)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4885..5340
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4972..4991)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5182..5203
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5418..5522
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5523..5591,5592..6383)
                     /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     complement(5741..5760)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat ttaggtgaca
     1021 ctatagaata ctcaagctat gcatccaatt tgagagacct gtgcggggct cgaatccccc
     1081 cattttcttc ttttacatca tccccccttc ctaaccaacc tgcaggcgca cctccctgct
     1141 gtgccatcag attactccgg gaacctttcc acggaaaaga cacgagacaa gggaaaagcc
     1201 aactaaatgg gcactcgagc tattcctttg ccctcggacg agtgctgggg cgtcggtttc
     1261 cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt ctgcgggcga
     1321 tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat cgaccctgcg
     1381 cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg tcaagaccaa
     1441 tgcggagcat atacgcccgg agccgaggcg atcctgcaag ctccggatgc ctccgctcga
     1501 agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag atgttggcga
     1561 cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct gttatgcggc
     1621 cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc cggacttcgg
     1681 ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac gcactgacgg
     1741 tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg catataaaat
     1801 cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac ccgctcgtct
     1861 ggctaagatc ggccgcagcg atcgcatcca ttgcctccgc gaccggctgc agaacagcgg
     1921 gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg gagatgcaat
     1981 aggtcaggct ctcgctaaat tccccaatgt caagcacttc cggaatcggg agcgcggccg
     2041 atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag ctatttaccc
     2101 gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct tcgccctccg
     2161 agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc tcgacagacg
     2221 tcgcggtgag ttcaggcttt ttcatgctag ctgatgcttt attgctttct cttaaatttg
     2281 ggataaccgt agtttatgcg gcagtagttt gacacctttt cgagggggaa agagacaaaa
     2341 aaggagaaga caaggattaa agggtgcacc caaaggagga aacacacggt tggtgtgcag
     2401 tgatgaatat caagttgcac aacagtgcag actcaaaacg gcactaatga accagatgtc
     2461 agacatgcag ctacgatcta cacaaacagt gactctctaa tgccgaagga taggcaccgc
     2521 agtgcacgta cacagcggca tagattgaaa tactacataa aggttataca ttgattataa
     2581 tgaaagacat ttaagcgcag cggagctctt aactacccga tcctgatcca gatcctgatc
     2641 ccgcgtagtc agggacatca tacgggtaac tgccggcgta gtccggtaca tcatatggat
     2701 atgatcccgc ataatccgga acatcgtagg ggtaggatcc cttgctgatt cgacgaaaca
     2761 catggtaata gaggtcccaa gcctgcagga gatccttaac gttcccagac ttcatgtatt
     2821 ttctacacca ctcttgtgct tccattaggt cgcgcccgta agcctggttg aagctcgtct
     2881 ccttcaacgt ctgtggacca cgctccatca tagcgtggag cggctccaac acttcaaaca
     2941 tacccttcac gttgcgctca ccgaaatata gtcgggacgc ttcttccaat ccctcgtgcc
     3001 acatctcgtg ccagagtata agcttagcat aatcgggtac gtcgtaagga tacgaacctg
     3061 cataatctgg aacgtcataa gggtacgaac ctgcgtaatc tggaacgtcg tatgggtaag
     3121 aaccggaacc ggaaccacta ccagaaccca taggtacccg ttgcagttga gtttctgttt
     3181 tcgggcttgt tggtatactt gtttgattaa tgtggctgat agctaagcta tcgcaaatat
     3241 ttgtacaaga catgaggggg caaaaaaata agccaagtgt atttgttagg cttccgtgcc
     3301 tgttagagaa atatgtaagg aaacgcctgc gctacacagc ttgagcaacg cgtggggctc
     3361 gaatcccccc attttcttct tttacatcat ccccccttcc taaccaacct gcaggcgcac
     3421 ctccctgctg tgccatcaga ttactccggg aacctttcca cggaaaagac acgagacaag
     3481 ggaaaggcca actaaatggg caccatggct attcctttgc cctcggacga gtgctggggc
     3541 gtcggtttcc actatcggcg agtacttcta cacagccatc ggtccagacg gccgcgcttc
     3601 tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc
     3661 gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac caagctctga tagagttggt
     3721 caagaccaat gcggagcata tacgcccgga gccgaggcga tcctgcaagc tccggatgcc
     3781 tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa ccacggcctc cagaagaaga
     3841 tgttggcgac ctcgtattgg gaatccccga acatcgcctc gctccagtca atgaccgctg
     3901 ttatgcggcc attgtccgtc aggacattgt tggagccgaa atccgcgtgc acgaggtgcc
     3961 ggacttcggg gcagtcctcg gcccaaagca tcagctcatc gagagcctgc gcgacggacg
     4021 cactgacggt gtcgtccatc acagtttgcc agtgatacac atggggatca gcaatcgcgc
     4081 atataaaatc acgccatgta gtgtattgac cgattccttg cggtccgaat gggccgaacc
     4141 cgctcgtctg gctaagatcg gccgcagcga tcgcatccat tgcctccgcg accggctgca
     4201 gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt gacaccctgt gcacggcggg
     4261 agatgcaata ggtcaggctc tcgctaaatt ccccaatgtc aagcacttcc ggaatcggga
     4321 gcgcggccga tgcaaagtgc cgataaacat aacgatcttt gtagaaacca tcggcgcagc
     4381 tatttacccg caggacatat ccacgccctc ctacatcgaa gctgaaagca cgagattctt
     4441 cgccctccga gagctgcatc aggtcggaga cgctgtcgaa cttttcgatc agaaacttct
     4501 cgacagacgt cgcggtgagt tcaggctttt tcatgaattc cactagagct tattttatgg
     4561 cagcaacgag accttacgta aagcaaaact aaataagagc ggagactgca atgcagagta
     4621 aaaaaaaaaa aaacatgaat ttcagaagac cttgctgtgc ccctagcttg cagcaggtct
     4681 gcattatacg ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
     4741 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     4801 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     4861 ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagt gcggcgggtg
     4921 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     4981 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5041 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5101 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5161 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5221 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5281 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     5341 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tcaggtggca
     5401 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5461 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5521 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5581 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5641 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5701 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5761 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5821 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5881 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5941 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6001 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6061 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6121 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6181 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6241 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtaattatct
     6301 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6361 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6421 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6481 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6541 tcaaaggatc ttc
//
