LOCUS       sequence-355743-        5536 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 5536)
  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 5536)
  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-eYFP
FEATURES             Location/Qualifiers
     source          1..5536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(960..962,963..1673)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1002..1023)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1263..1282)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1610..1631
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2661..2679)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2662..2679)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2697..2713)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2697..2713)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2710..2732)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2721..2737
                     /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(2745..2751,2752..2769,2770..2775))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2790..2811
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2928..2945)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3099..3687)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3179..3198)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3858..4649,4650..4718))
                     /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     4481..4500
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4719..4823)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4901..5356)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5038..5059)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5250..5269
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5482..5504
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5496..5513
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5497..5513
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5520..5536,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5520..5536,1..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 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1021 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1081 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1141 tgaccacctt cggctacggc ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc
     1201 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1261 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1321 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1381 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     1441 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1501 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1561 tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1621 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggatccg
     1681 gatcaggatc tggatcagga tcgggtagtt aagagctccg ctgcgcttaa atgtctttca
     1741 ttataatcaa tgtataacct ttatgtagta tttcaatcta tgccgctgtg tacgtgcact
     1801 gcggtgccta tccttcggca ttagagagtc actgtttgtg tagatcgtag ctgcatgtct
     1861 gacatctggt tcattagtgc cgttttgagt ctgcactgtt gtgcaacttg atattcatca
     1921 ctgcacacca accgtgtgtt tcctcctttg ggtgcaccct ttaatccttg tcttctcctt
     1981 ttttgtctct ttccccctcg aaaaggtgtc aaactactgc cgcataaact acggttatcc
     2041 caaatttaag agaaagcaat aaagcatcag ctagcatgcc tttgtctcaa gaagaatcca
     2101 ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg
     2161 tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt
     2221 ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg
     2281 gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg
     2341 gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca
     2401 gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg
     2461 agggctaact cgagtgccca tttagttggc ttttcccttg tctcgtgtct tttccgtgga
     2521 aaggttcccg gagtaatctg atggcacagc agggaggtgc gcctgcaggt tggttaggaa
     2581 ggggggatga tgtaaaagaa gaaaatgggg ggattcgagc cccgcacagg tctctcaaat
     2641 tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta atcatggtca
     2701 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     2761 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     2821 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     2881 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2941 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3001 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3061 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3121 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3181 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3241 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3301 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3361 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3421 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3481 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3541 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3601 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3661 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3721 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3781 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3841 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3901 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataattac gatacgggag
     3961 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4021 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4081 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4141 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4201 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4261 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4321 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4381 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4441 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4501 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4561 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4621 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4681 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4741 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4801 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga tgcggtgtga
     4861 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt
     4921 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     4981 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     5041 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     5101 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     5161 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     5221 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     5281 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc acttaatgcg
     5341 ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     5401 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     5461 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt
     5521 aatacgactc actata
//
