LOCUS       sequence-441446-        5686 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 5686)
  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 5686)
  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: pPOTvGPI(EP)-blast-mNG
FEATURES             Location/Qualifiers
     source          1..5686
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2716..2734)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2717..2734)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2752..2768)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2752..2768)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2765..2787)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2776..2792
                     /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(2800..2806,2807..2824,2825..2830))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2845..2866
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2983..3000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3154..3742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3234..3253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3913..4704,4705..4773))
                     /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     4536..4555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4774..4878)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4956..5411)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5093..5114)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5305..5324
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5537..5559
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5551..5568
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5552..5568
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5575..5594
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5575..5593
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gcagtctccg ctcttattta gttttgcttt acgtaaggtc tcgttgctgc cataaaataa
       61 gctctagtgg aattcatgcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
      121 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
      181 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
      241 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
      301 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
      361 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
      421 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaact cgagtgccca
      481 tttagttggc ttttcccttg tctcgtgtct tttccgtgga aaggttcccg gagtaatctg
      541 atggcacagc agggaggtgc gcctgcaggt tggttaggaa ggggggatga tgtaaaagaa
      601 gaaaatgggg ggattcgagc cccacgcgtt gctcaagctg tgtagcgcag gcgtttcctt
      661 acatatttct ctaacaggca cggaagccta acaaatacac ttggcttatt tttttgcccc
      721 ctcatgtctt gtacaaatat ttgcgatagc ttagctatca gccacattaa tcaaacaagt
      781 ataccaacaa gcccgaaaac agaaactcaa ctgcaacggg tacctatggc accacgctct
      841 ctttatcttc tggccatact cctgttttca gccaaccttt tcgcaggtgt gggctttgca
      901 gctgccgcgg agggaggttc tggtagtggt tccggttccg gttctaagct tgtctcgaaa
      961 ggtgaggaag acaatatggc cagcttgcca gcaacgcacg agcttcatat atttgggtcg
     1021 atcaacggtg tggatttcga catggtgggt caaggcacag gtaatcctaa tgacgggtat
     1081 gaagaactca atttgaagag cactaaggga gatctccagt tttccccgtg gattcttgtg
     1141 ccgcatattg gttatggttt tcatcagtac ttaccatacc ccgatggaat gtcgcccttc
     1201 caagcagcta tggtggacgg ctcggggtat caagtacatc gcaccatgca atttgaggac
     1261 ggagcatccc tcaccgtaaa ctaccgctat acgtatgagg ggagccacat aaaaggtgag
     1321 gctcaagtta aaggcacagg cttcccagct gacggcccgg tcatgaccaa ttcattaacc
     1381 gctgctgatt ggtgccgttc taagaagacc taccctaacg acaagacgat cattagtact
     1441 tttaagtgga gttataccac aggaaacgga aagcggtatc gaagcactgc tcgaacaacc
     1501 tacacatttg cgaagccaat ggccgcgaac tacttgaaga accagccaat gtatgtgttt
     1561 cgaaaaacgg aactgaagca ttcgaaaacc gagcttaatt ttaaagagtg gcagaaggcg
     1621 tttacagacg tgatggggat ggacgaattg tataagggat ccggatcagg atctggatca
     1681 ggatcgggta gtcccggcgc ggcaactctg aagtccgttg cgctaccgtt tgccgtagcg
     1741 gccgcggcac tagtagctgc attctaggag ctccgctgcg cttaaatgtc tttcattata
     1801 atcaatgtat aacctttatg tagtatttca atctatgccg ctgtgtacgt gcactgcggt
     1861 gcctatcctt cggcattaga gagtcactgt ttgtgtagat cgtagctgca tgtctgacat
     1921 ctggttcatt agtgccgttt tgagtctgca ctgttgtgca acttgatatt catcactgca
     1981 caccaaccgt gtgtttcctc ctttgggtgc accctttaat ccttgtcttc tccttttttg
     2041 tctctttccc cctcgaaaag gtgtcaaact actgccgcat aaactacggt tatcccaaat
     2101 ttaagagaaa gcaataaagc atcagctagc atgcctttgt ctcaagaaga atccaccctc
     2161 attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc
     2221 agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact
     2281 gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac
     2341 ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg
     2401 tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat
     2461 ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc
     2521 taactcgagt gcccatttag ttggcttttc ccttgtctcg tgtcttttcc gtggaaaggt
     2581 tcccggagta atctgatggc acagcaggga ggtgcgcctg caggttggtt aggaaggggg
     2641 gatgatgtaa aagaagaaaa tggggggatt cgagccccgc acaggtctct caaattggat
     2701 gcatagcttg agtattctat agtgtcacct aaatagcttg gcgtaatcat ggtcatagct
     2761 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     2821 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     2881 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2941 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3001 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3061 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3121 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3181 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3241 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3301 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3361 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3421 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3481 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3541 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3601 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3661 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3721 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3781 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3841 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3901 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3961 tcgttcatcc atagttgcct gactccccgt cgtgtagata attacgatac gggagggctt
     4021 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4081 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4141 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4201 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4261 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4321 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4381 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4441 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4501 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4561 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4621 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4681 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4741 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4801 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4861 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac
     4921 cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt
     4981 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     5041 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     5101 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     5161 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     5221 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     5281 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     5341 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcactta atgcgccgct
     5401 acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     5461 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     5521 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac
     5581 gactcactat agggcgaatt gggcccgtat aatgcagacc tgctgcaagc taggggcaca
     5641 gcaaggtctt ctgaaattca tgtttttttt ttttttactc tgcatt
//
