LOCUS       sequence-355575-        5296 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 5296)
  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 5296)
  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-10V5
FEATURES             Location/Qualifiers
     source          1..5296
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             960..1001
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1008..1049
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1056..1097
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1104..1145
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1152..1193
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1200..1241
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1248..1289
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1296..1337
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1344..1385
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1392..1433
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     promoter        complement(2421..2439)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2422..2439)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2457..2473)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2457..2473)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2470..2492)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2481..2497
                     /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(2505..2511,2512..2529,2530..2535))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2550..2571
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2688..2705)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2859..3447)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2939..2958)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3618..4409,4410..4478))
                     /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     4241..4260
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4479..4583)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4661..5116)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4798..4819)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5010..5029
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5242..5264
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5256..5273
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5257..5273
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5280..5296,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5280..5296,1..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 gaaagccaat tccaaaccct cttcttggac tcgactccac cggctctggt aaaccgatcc
     1021 cgaatccact ccttggattg gattctaccg gatcggggaa accgatacct aatccgctgc
     1081 tgggccttga ctcgacgggc tcgggaaaac caatacctaa tccattgctt ggactcgact
     1141 cgactggcag tggtaaaccc ataccgaatc ccttgctggg gctcgactcc acagggtccg
     1201 ggaagcctat acccaatccg ctcctgggtt tggattctac tggcagtggg aagcccattc
     1261 cgaatcctct gctcggcttg gacagtacag gctcgggaaa gcccatcccc aatccgctgt
     1321 tggggcttga ttcaactggg tcgggtaaac ccatcccgaa tccgcttctc gggctcgatt
     1381 caactgggtc tggaaagccc atccctaacc cgcttctcgg tctggacagc acgggatccg
     1441 gatcaggatc tggatcagga tcgggtagtt aagagctccg ctgcgcttaa atgtctttca
     1501 ttataatcaa tgtataacct ttatgtagta tttcaatcta tgccgctgtg tacgtgcact
     1561 gcggtgccta tccttcggca ttagagagtc actgtttgtg tagatcgtag ctgcatgtct
     1621 gacatctggt tcattagtgc cgttttgagt ctgcactgtt gtgcaacttg atattcatca
     1681 ctgcacacca accgtgtgtt tcctcctttg ggtgcaccct ttaatccttg tcttctcctt
     1741 ttttgtctct ttccccctcg aaaaggtgtc aaactactgc cgcataaact acggttatcc
     1801 caaatttaag agaaagcaat aaagcatcag ctagcatgcc tttgtctcaa gaagaatcca
     1861 ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg
     1921 tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt
     1981 ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg
     2041 gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg
     2101 gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca
     2161 gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg
     2221 agggctaact cgagtgccca tttagttggc ttttcccttg tctcgtgtct tttccgtgga
     2281 aaggttcccg gagtaatctg atggcacagc agggaggtgc gcctgcaggt tggttaggaa
     2341 ggggggatga tgtaaaagaa gaaaatgggg ggattcgagc cccgcacagg tctctcaaat
     2401 tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta atcatggtca
     2461 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     2521 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     2581 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     2641 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2701 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2761 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2821 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2881 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2941 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3001 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3061 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3121 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3181 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3241 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3301 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3361 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3421 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3481 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3541 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3601 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3661 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataattac gatacgggag
     3721 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3781 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3841 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3901 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3961 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4021 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4081 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4141 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4201 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4261 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4321 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4381 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4441 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4501 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4561 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga tgcggtgtga
     4621 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt
     4681 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     4741 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     4801 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4861 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     4921 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4981 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     5041 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc acttaatgcg
     5101 ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     5161 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     5221 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt
     5281 aatacgactc actata
//
