LOCUS       sequence-495182-        7231 bp    DNA     circular SYN 11-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 7231)
  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 7231)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 11, 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::tbTurboID::3HA
FEATURES             Location/Qualifiers
     source          1..7231
                     /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(3704..3730)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3737..3763)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3770..3796)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4187..5212)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4187..5212)
                     /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(5380..5399)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5381..5399)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5406..5423)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5406..5422)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5415..5437)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5563..6018
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5650..5669)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5860..5881
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6096..6200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6201..6269,6270..7061)
                     /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(6419..6438)
                     /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 cttctcggca gacctaaggg
     2761 agatctcgcc gcccatccaa ggtttgataa caccgtcttg ttccaacaac aatgctccct
     2821 gtttgtctat gcctcgtgaa atgccaaaaa tttctttatc tccaataata agtttcaccg
     2881 gtcggttgat aaagttatct aacttttccc agcggggaag gtagggtgcg aggccttcct
     2941 gttcgaataa ctccaaggcg gcccgaagtt ccctaatgag ggttgccgcc agcgtattcc
     3001 gatccaggtt tattccggct tcctgcaggg ttatccaacc ctgattgacc acgctctcct
     3061 cgaccctgcg catcgccacg ttaatacctg caccaatcac gatttgtgcg gcatcgcccg
     3121 ttataccggc taattcgaca agaatacccg ccaatttacg atcttgtaaa tacagatcgt
     3181 tgggccattt gaccctgact ttatctgccc caagtttgcg tagtgcttcc gccattacaa
     3241 tgccaattac tggacctaga cctattgccg ccggacctct cttaagtctc cagaacatgc
     3301 tcagataaag gttcgccccg aatggactaa accactttcg accccgggac ccgcgtccgg
     3361 cctgctgata ttcagcaatg caagcatcac ctgacttgag ttcacctatg cgatctaaca
     3421 ggtattggtt agtggaatca accacgggga ggacagccac actgccgcca tcgagctgcc
     3481 ctaggatctg ctttgcgttt aaaagaggaa ttggctctgg tagcgaatac ccttttccag
     3541 gcacagtaaa cacatccact ccccaatcac gtaacgtctg aatgtgctta ttaatagcag
     3601 cacgcgacat gccgagggtt tctcccaact gttcaccact gtgaaattcc ccattagcaa
     3661 ggagcgcgat aagttttagt ggcaccgtgt tatctttaag cttagcataa tcgggtacgt
     3721 cgtaaggata cgaacctgca taatctggaa cgtcataagg gtacgaacct gcgtaatctg
     3781 gaacgtcgta tgggtaagaa ccggaaccgg aaccactacc agaacccata ggtacccgtt
     3841 gcagttgagt ttctgttttc gggcttgttg gtatacttgt ttgattaatg tggctgatag
     3901 ctaagctatc gcaaatattt gtacaagaca tgagggggca aaaaaataag ccaagtgtat
     3961 ttgttaggct tccgtgcctg ttagagaaat atgtaaggaa acgcctgcgc tacacagctt
     4021 gagcaacgcg tggggctcga atccccccat tttcttcttt tacatcatcc ccccttccta
     4081 accaacctgc aggcgcacct ccctgctgtg ccatcagatt actccgggaa cctttccacg
     4141 gaaaagacac gagacaaggg aaaggccaac taaatgggca ccatggctat tcctttgccc
     4201 tcggacgagt gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg
     4261 tccagacggc cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc
     4321 ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca
     4381 agctctgata gagttggtca agaccaatgc ggagcatata cgcccggagc cgaggcgatc
     4441 ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc
     4501 acggcctcca gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc
     4561 tccagtcaat gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat
     4621 ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga
     4681 gagcctgcgc gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat
     4741 ggggatcagc aatcgcgcat ataaaatcac gccatgtagt gtattgaccg attccttgcg
     4801 gtccgaatgg gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccattg
     4861 cctccgcgac cggctgcaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga
     4921 caccctgtgc acggcgggag atgcaatagg tcaggctctc gctaaattcc ccaatgtcaa
     4981 gcacttccgg aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt
     5041 agaaaccatc ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc
     5101 tgaaagcacg agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact
     5161 tttcgatcag aaacttctcg acagacgtcg cggtgagttc aggctttttc atgaattcca
     5221 ctagagctta ttttatggca gcaacgagac cttacgtaaa gcaaaactaa ataagagcgg
     5281 agactgcaat gcagagtaaa aaaaaaaaaa acatgaattt cagaagacct tgctgtgccc
     5341 ctagcttgca gcaggtctgc attatacggg cccaattcgc cctatagtga gtcgtattac
     5401 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     5461 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     5521 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg
     5581 cattaagtgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     5641 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5701 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5761 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5821 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5881 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5941 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     6001 tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     6061 acaccgcatc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     6121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     6181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     6241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     6301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6961 cccgtatcgt aattatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     7021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     7081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     7141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     7201 cagaccccgt agaaaagatc aaaggatctt c
//
