LOCUS       sequence-355741-        5368 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 5368)
  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 5368)
  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-CLIPtag
FEATURES             Location/Qualifiers
     source          1..5368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             960..1505
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylcytosine 
                     derivatives"
                     /label=CLIP-tag(TM)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLA
                     VKEWLLAHEGHRLGKPGLG"
     promoter        complement(2493..2511)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2494..2511)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2529..2545)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2529..2545)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2542..2564)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2553..2569
                     /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(2577..2583,2584..2601,2602..2607))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2622..2643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2760..2777)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2931..3519)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3011..3030)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3690..4481,4482..4550))
                     /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     4313..4332
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4551..4655)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4733..5188)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4870..4891)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5082..5101
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5314..5336
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5328..5345
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5329..5345
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5352..5368,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5352..5368,1..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 tctaagctta
      961 tggacaaaga ctgcgaaatg aagcgcacca ccctggatag ccctctgggc aagctggaac
     1021 tgtctgggtg cgaacagggc ctgcaccgta tcatcttcct gggcaaagga acatctgccg
     1081 ccgacgccgt ggaagtgcct gccccagccg ccgtgctggg cggaccagag ccactgatcc
     1141 aggccaccgc ctggctcaac gcctactttc accagcctga ggccatcgag gagttccctg
     1201 tgccagccct gcaccaccca gtgttccagc aggagagctt tacccgccag gtgctgtgga
     1261 aactgctgaa agtggtgaag ttcggagagg tcatcagcga gagccacctg gccgccctgg
     1321 tgggcaatcc cgccgccacc gccgccgtga acaccgccct ggacggaaat cccgtgccca
     1381 ttctgatccc ctgccaccgg gtggtgcagg gcgacagcga cgtggggccc tacctgggcg
     1441 ggctcgccgt gaaagagtgg ctgctggccc acgagggcca cagactgggc aagcctgggc
     1501 tgggtggatc cggatcagga tctggatcag gatcgggtag ttaagagctc cgctgcgctt
     1561 aaatgtcttt cattataatc aatgtataac ctttatgtag tatttcaatc tatgccgctg
     1621 tgtacgtgca ctgcggtgcc tatccttcgg cattagagag tcactgtttg tgtagatcgt
     1681 agctgcatgt ctgacatctg gttcattagt gccgttttga gtctgcactg ttgtgcaact
     1741 tgatattcat cactgcacac caaccgtgtg tttcctcctt tgggtgcacc ctttaatcct
     1801 tgtcttctcc ttttttgtct ctttccccct cgaaaaggtg tcaaactact gccgcataaa
     1861 ctacggttat cccaaattta agagaaagca ataaagcatc agctagcatg cctttgtctc
     1921 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg
     1981 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca
     2041 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg
     2101 ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct
     2161 tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca
     2221 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg
     2281 gttatgtgtg ggagggctaa ctcgagtgcc catttagttg gcttttccct tgtctcgtgt
     2341 cttttccgtg gaaaggttcc cggagtaatc tgatggcaca gcagggaggt gcgcctgcag
     2401 gttggttagg aaggggggat gatgtaaaag aagaaaatgg ggggattcga gccccgcaca
     2461 ggtctctcaa attggatgca tagcttgagt attctatagt gtcacctaaa tagcttggcg
     2521 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     2581 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     2641 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2701 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2761 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2821 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2881 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2941 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3001 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3061 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3121 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3181 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3241 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3301 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3361 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3421 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3481 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3541 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3601 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3661 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3721 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataatt
     3781 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3841 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3901 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3961 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4021 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4081 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4141 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4201 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4261 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4321 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4381 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4441 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4501 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4561 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4621 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4681 gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta
     4741 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     4801 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     4861 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     4921 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     4981 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     5041 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     5101 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     5161 gcacttaatg cgccgctaca gggcgcgtcc attcgccatt caggctgcgc aactgttggg
     5221 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     5281 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     5341 ccagtgaatt gtaatacgac tcactata
//
