LOCUS       sequence-355420-        7528 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 7528)
  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 7528)
  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-blast-3HaloTag
FEATURES             Location/Qualifiers
     source          1..7528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             981..1868
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     CDS             1878..2765
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     CDS             2778..3665
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     promoter        complement(4653..4671)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4654..4671)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4689..4705)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4689..4705)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4702..4724)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4713..4729
                     /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(4737..4743,4744..4761,4762..4767))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4782..4803
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4920..4937)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5091..5679)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5171..5190)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5850..6641,6642..6710))
                     /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     6473..6492
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6711..6815)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6893..7348)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7030..7051)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7242..7261
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7474..7496
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7488..7505
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7489..7505
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(7512..7528,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(7512..7528,1..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 atgcctttgt
      181 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct
      241 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg
      301 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg
      361 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca
      421 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga cctgcatcct gggatcaaag
      481 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct
      541 ctggttatgt gtgggagggc taaccatggt gcccatttag ttggcctttc ccttgtctcg
      601 tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga ggtgcgcctg
      661 caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt cgagccccac
      721 gcgttgctca agctgtgtag cgcaggcgtt tccttacata tttctctaac aggcacggaa
      781 gcctaacaaa tacacttggc ttattttttt gccccctcat gtcttgtaca aatatttgcg
      841 atagcttagc tatcagccac attaatcaaa caagtatacc aacaagcccg aaaacagaaa
      901 ctcaactgca acgggtacct atgggttctg gtagtggttc cggttccggt tctaagcttg
      961 gtgggtctgg aagcggtagt gccgaaatag gcacaggctt cccctttgat ccgcattacg
     1021 tggaagttct gggggagcgt atgcactacg tcgacgttgg acccagggat ggtacgccgg
     1081 ttcttttcct gcacgggaac cccacgtcga gctacgtgtg gcgaaacatc ataccgcacg
     1141 tggctcccac gcaccgatgt attgcgccag atcttatagg catggggaag tccgacaaac
     1201 ccgatcttgg ttacttcttc gacgaccatg tgcgcttcat ggatgctttt atcgaggcac
     1261 ttggattgga ggaggttgtt ttggtaatac acgactgggg ctcagccctt ggctttcatt
     1321 gggcgaagcg taatccagag cgggttaaag gcatcgcttt tatggagttc ataaggccca
     1381 tcccgacgtg ggatgagtgg ccagagtttg ccagggagac cttccaagcc tttcgcacga
     1441 ccgacgtcgg tcgcaaattg ataatagacc aaaacgtgtt tattgaggga accctgccta
     1501 tgggagtagt caggcccctc actgaagtgg aaatggacca ctatagggag ccatttttga
     1561 atccggttga tcgggaacct ctttggcgat ttccaaatga acttcctatt gcaggagagc
     1621 ccgccaacat cgttgctctg gtcgaggaat atatggattg gctccatcag tcaccggtcc
     1681 cgaaacttct gttttggggg accccaggag ttttgatacc ccctgccgag gcggcccgcc
     1741 ttgccaaatc gcttcccaat tgcaaagcag ttgacatcgg accaggtctc aacctgctcc
     1801 aggaagataa tccagatttg atcggctcgg aaatcgctcg ctggctgtcg acactggaga
     1861 tatctggttc gggctcggct gagataggta ctgggtttcc gttcgaccct cactacgtag
     1921 aagttcttgg agagcggatg cactatgtcg atgtggggcc tcgcgatggc acccccgtac
     1981 ttttcctcca cggcaacccc acgtcgagct atgtctggag gaacataata ccacacgtgg
     2041 ctccaacgca taggtgtata gctcccgacc ttataggaat gggcaaaagc gacaaacccg
     2101 accttggata cttcttcgat gatcacgtac gcttcatgga cgctttcatc gaagccctgg
     2161 gcttggagga ggttgtcttg gttattcatg attggggatc agctcttggg tttcactggg
     2221 ctaaacgcaa ccctgaacgg gtaaaaggca tcgcgttcat ggagtttata cgtcccatac
     2281 caacctggga cgaatggccg gagttcgcac gtgagacctt ccaggccttt cgcactaccg
     2341 atgtaggcag gaagctgatc attgaccaaa acgtctttat tgaaggcacc ctgcctatgg
     2401 gagttgtcag gcctcttact gaggtcgaaa tggaccatta ccgggaaccg tttctgaatc
     2461 cggtggatcg ggaaccactt tggaggttcc ccaatgaact cccgattgcc ggcgagccag
     2521 ccaacatcgt tgcgctcgtt gaagaataca tggactggct gcaccagagt ccggtgccga
     2581 aactcttgtt ttggggcacc cctggcgttc tgatcccacc ggccgaggcg gcccgactcg
     2641 ccaagtcatt gcctaactgt aaagcggttg atatcggccc tggacttaat cttctgcaag
     2701 aagacaaccc agacctgatc ggctccgaga tcgcacggtg gttgtctact cttgaaattt
     2761 ccggttcgcc cgggtcggct gagattggta cggggtttcc ttttgacccg cactacgttg
     2821 aggtgttggg tgaacgtatg cattatgttg atgttggtcc ccgtgatggt acgcccgtgc
     2881 tgtttttgca tggtaacccc acgtcaagct atgtgtggag gaacattatt ccccacgtgg
     2941 cacctactca tcgctgtatt gctccagacc ttattggtat gggtaagtca gataaaccgg
     3001 atcttggtta cttctttgac gatcacgtgc gttttatgga tgcatttatt gaagcgcttg
     3061 gccttgaaga agttgtgtta gtgattcacg attggggttc tgccttggga tttcattggg
     3121 ctaaacgtaa tcccgaaaga gttaaaggta ttgcattcat ggagttcatt cgtcctattc
     3181 ctacgtggga tgagtggcca gagtttgcgc gcgagacgtt tcaagcgttt cgtacaacgg
     3241 acgtgggtcg taagctgatt attgatcaaa atgtttttat tgagggtacg ctgccgatgg
     3301 gtgttgtgcg tcccctgact gaagtggaga tggatcacta ccgtgagcca tttttaaatc
     3361 cagtggaccg tgagccattg tggcgcttcc ctaacgagct gcccattgct ggtgaaccgg
     3421 ctaacattgt ggcgcttgtg gaggagtata tggattggct tcatcaatca cccgtgccta
     3481 agttgctttt ttggggtacg ccaggtgtgc ttattccacc tgctgaagca gcacgcttgg
     3541 ctaaatcact tccaaactgc aaagcagttg atattggtcc gggtctgaac ctgttgcaag
     3601 aagataatcc cgatcttatt ggttccgaga ttgcccgttg gctttcgaca cttgaaattt
     3661 ctggtggatc cggatcagga tctggatcag gatcgggtag ttaagagctc cgctgcgctt
     3721 aaatgtcttt cattataatc aatgtataac ctttatgtag tatttcaatc tatgccgctg
     3781 tgtacgtgca ctgcggtgcc tatccttcgg cattagagag tcactgtttg tgtagatcgt
     3841 agctgcatgt ctgacatctg gttcattagt gccgttttga gtctgcactg ttgtgcaact
     3901 tgatattcat cactgcacac caaccgtgtg tttcctcctt tgggtgcacc ctttaatcct
     3961 tgtcttctcc ttttttgtct ctttccccct cgaaaaggtg tcaaactact gccgcataaa
     4021 ctacggttat cccaaattta agagaaagca ataaagcatc agctagcatg cctttgtctc
     4081 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg
     4141 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca
     4201 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg
     4261 ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct
     4321 tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca
     4381 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg
     4441 gttatgtgtg ggagggctaa ctcgagtgcc catttagttg gcttttccct tgtctcgtgt
     4501 cttttccgtg gaaaggttcc cggagtaatc tgatggcaca gcagggaggt gcgcctgcag
     4561 gttggttagg aaggggggat gatgtaaaag aagaaaatgg ggggattcga gccccgcaca
     4621 ggtctctcaa attggatgca tagcttgagt attctatagt gtcacctaaa tagcttggcg
     4681 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4741 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4801 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4861 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4921 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4981 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5041 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5101 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5161 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5221 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5281 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5341 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5401 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5461 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5521 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5581 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5641 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5701 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5761 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5821 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5881 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataatt
     5941 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6001 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6061 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6121 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6181 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6241 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6301 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6361 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6421 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6481 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6541 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6601 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6661 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6721 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6781 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6841 gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta
     6901 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     6961 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     7021 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     7081 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     7141 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     7201 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     7261 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     7321 gcacttaatg cgccgctaca gggcgcgtcc attcgccatt caggctgcgc aactgttggg
     7381 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     7441 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     7501 ccagtgaatt gtaatacgac tcactata
//
