LOCUS       sequence-374847-        6778 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 6778)
  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 6778)
  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-hygro-BFP
FEATURES             Location/Qualifiers
     source          1..6778
                     /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             2685..3710
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2685..3710
                     /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(3903..3921)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3904..3921)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3939..3955)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3939..3955)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3952..3974)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3963..3979
                     /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(3987..3993,3994..4011,4012..4017))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4032..4053
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4170..4187)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4341..4929)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4421..4440)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5100..5891,5892..5960))
                     /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     5723..5742
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5961..6065)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6143..6598)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6280..6301)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6492..6511
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6724..6746
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6738..6755
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6739..6755
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6762..6778,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6762..6778,1..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 tagttggcct 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 ggttctaagc ttagtgagtt gattaaggaa aacatgcaca
     1621 tgaagttgta catggaagga acagtagata atcatcattt caaatgtacg tcagaaggtg
     1681 agggcaaacc ttatgaagga actcaaacta tgagaattaa agtcgttgag ggtggcccac
     1741 taccatttgc attcgatatc ctcgcaacat cttttctgta tggttctaaa acctttatca
     1801 accatacaca aggcatacca gatttcttta agcaatcatt tcctgaaggt ttcacatggg
     1861 aaagagtaac tacctatgaa gatggtggtg tccttacagc tactcaagac acgtctttgc
     1921 aggatggttg tttaatctac aatgttaaga taagaggagt gaactttaca tccaatggac
     1981 cagtcatgca aaagaaaact ctaggttggg aggcattcac agaaactttg tacccagccg
     2041 atgggggctt agaagggaga aatgacatgg ctttaaaact ggttggcggt agccatttga
     2101 ttgcgaacgc taaaacaacc tacagaagca aaaagccagc taaaaatctt aagatgcctg
     2161 tatactacgt ggattacaga ttagaaagga aggaagctaa taatgaaact tacgttgaac
     2221 agcacgaggt ggccgttgcc cgttattgcg accttccttc aaaattggga cacaaactaa
     2281 acggatccgg atcaggatct ggatcaggat cgggtagtta agagctccgc tgcgcttaaa
     2341 tgtctttcat tataatcaat gtataacctt tatgtagtat ttcaatctat gccgctgtgt
     2401 acgtgcactg cggtgcctat ccttcggcat tagagagtca ctgtttgtgt agatcgtagc
     2461 tgcatgtctg acatctggtt cattagtgcc gttttgagtc tgcactgttg tgcaacttga
     2521 tattcatcac tgcacaccaa ccgtgtgttt cctcctttgg gtgcaccctt taatccttgt
     2581 cttctccttt tttgtctctt tccccctcga aaaggtgtca aactactgcc gcataaacta
     2641 cggttatccc aaatttaaga gaaagcaata aagcatcagc tagcatgaaa aagcctgaac
     2701 tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga
     2761 tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat
     2821 atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc
     2881 actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa tttagcgaga
     2941 gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa
     3001 ccgaactgcc cgctgttctg cagccggtcg cggaggcaat ggatgcgatc gctgcggccg
     3061 atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta
     3121 catggcgtga ttttatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga
     3181 tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg
     3241 aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga
     3301 cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc
     3361 aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga
     3421 cgcgctactt cgagcggagg catccggagc ttgcaggatc gcctcggctc cgggcgtata
     3481 tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg
     3541 cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc
     3601 gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg
     3661 ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag ctcgagtgcc
     3721 catttagttg gcttttccct tgtctcgtgt cttttccgtg gaaaggttcc cggagtaatc
     3781 tgatggcaca gcagggaggt gcgcctgcag gttggttagg aaggggggat gatgtaaaag
     3841 aagaaaatgg ggggattcga gccccgcaca ggtctctcaa attggatgca tagcttgagt
     3901 attctatagt gtcacctaaa tagcttggcg taatcatggt catagctgtt tcctgtgtga
     3961 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4021 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4081 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4141 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4201 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4261 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4321 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4381 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4441 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4501 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4561 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4621 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4681 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4741 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4801 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4861 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4921 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4981 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5041 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5101 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5161 gttgcctgac tccccgtcgt gtagataatt acgatacggg agggcttacc atctggcccc
     5221 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5281 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5341 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5401 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5461 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5521 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5581 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5641 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5701 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5761 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5821 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5881 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5941 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6001 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6061 ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt gaaataccgc acagatgcgt
     6121 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta
     6181 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     6241 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     6301 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     6361 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     6421 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     6481 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     6541 gtcacgctgc gcgtaaccac cacacccgcc gcacttaatg cgccgctaca gggcgcgtcc
     6601 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     6661 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     6721 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactata
//
