LOCUS       sequence-441436-        4906 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 4906)
  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 4906)
  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-3FLAG
FEATURES             Location/Qualifiers
     source          1..4906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             875..898
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             905..928
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             935..958
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1946..1964)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1947..1964)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1982..1998)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1982..1998)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1995..2017)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2006..2022
                     /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(2030..2036,2037..2054,2055..2060))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2075..2096
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2213..2230)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2384..2972)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2464..2483)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3143..3934,3935..4003))
                     /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     3766..3785
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4004..4108)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4186..4641)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4323..4344)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4535..4554
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4767..4789
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4781..4798
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4782..4798
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4805..4824
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4805..4823
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 actctgcatt gcagtctccg ctcttattta gttttgcttt acgtaaggtc tcgttgctgc
       61 cataaaataa gctctagtgg aattcatgcc tttgtctcaa gaagaatcca ccctcattga
      121 aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc
      181 agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg
      241 accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac
      301 ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg
      361 acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca
      421 gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaact
      481 cgagtgccca tttagttggc ttttcccttg tctcgtgtct tttccgtgga aaggttcccg
      541 gagtaatctg atggcacagc agggaggtgc gcctgcaggt tggttaggaa ggggggatga
      601 tgtaaaagaa gaaaatgggg ggattcgagc cccacgcgtt gctcaagctg tgtagcgcag
      661 gcgtttcctt acatatttct ctaacaggca cggaagccta acaaatacac ttggcttatt
      721 tttttgcccc ctcatgtctt gtacaaatat ttgcgatagc ttagctatca gccacattaa
      781 tcaaacaagt ataccaacaa gcccgaaaac agaaactcaa ctgcaacggg tacctatggg
      841 ttctggtagt ggttccggtt ccggttctaa gcttgactac aaggacgacg acgataaagg
      901 gagtgactac aaagatgatg atgacaaggg ttcggattac aaagacgacg atgacaaggg
      961 atccggatca ggatctggat caggatcggg tagttaagag ctccgctgcg cttaaatgtc
     1021 tttcattata atcaatgtat aacctttatg tagtatttca atctatgccg ctgtgtacgt
     1081 gcactgcggt gcctatcctt cggcattaga gagtcactgt ttgtgtagat cgtagctgca
     1141 tgtctgacat ctggttcatt agtgccgttt tgagtctgca ctgttgtgca acttgatatt
     1201 catcactgca caccaaccgt gtgtttcctc ctttgggtgc accctttaat ccttgtcttc
     1261 tccttttttg tctctttccc cctcgaaaag gtgtcaaact actgccgcat aaactacggt
     1321 tatcccaaat ttaagagaaa gcaataaagc atcagctagc atgcctttgt ctcaagaaga
     1381 atccaccctc attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta
     1441 cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata
     1501 tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc
     1561 agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc
     1621 ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa
     1681 ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt
     1741 gtgggagggc taactcgagt gcccatttag ttggcttttc ccttgtctcg tgtcttttcc
     1801 gtggaaaggt tcccggagta atctgatggc acagcaggga ggtgcgcctg caggttggtt
     1861 aggaaggggg gatgatgtaa aagaagaaaa tggggggatt cgagccccgc acaggtctct
     1921 caaattggat gcatagcttg agtattctat agtgtcacct aaatagcttg gcgtaatcat
     1981 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     2041 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     2101 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     2161 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     2221 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2281 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2341 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2401 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2461 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2521 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2581 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2641 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2701 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2761 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2821 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2881 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2941 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3001 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3061 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3121 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3181 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata attacgatac
     3241 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3301 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3361 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3421 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3481 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3541 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     3601 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3661 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3721 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     3781 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     3841 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     3901 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     3961 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4021 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4081 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgatgcgg
     4141 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta
     4201 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     4261 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     4321 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     4381 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     4441 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     4501 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     4561 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcactta
     4621 atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg
     4681 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     4741 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
     4801 attgtaatac gactcactat agggcgaatt gggcccgtat aatgcagacc tgctgcaagc
     4861 taggggcaca gcaaggtctt ctgaaattca tgtttttttt tttttt
//
