LOCUS       sequence-355466-        5515 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 5515)
  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 5515)
  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-mScarlet
FEATURES             Location/Qualifiers
     source          1..5515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2640..2658)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2641..2658)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2676..2692)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2676..2692)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2689..2711)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2700..2716
                     /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(2724..2730,2731..2748,2749..2754))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2769..2790
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2907..2924)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3078..3666)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3158..3177)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3837..4628,4629..4697))
                     /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     4460..4479
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4698..4802)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4880..5335)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5017..5038)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5229..5248
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5461..5483
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5475..5492
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5476..5492
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5499..5515,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5499..5515,1..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 taagcaaagg ggaggctgtc ataaaggaat ttatgcgatt taaagtgcac atggaaggga
     1021 gtatgaacgg ccatgagttt gaaatagaag gagagggaga ggggcgccct tacgaaggta
     1081 ctcagacggc gaagctcaaa gtgactaagg gaggtcctct gccgtttagc tgggatatac
     1141 tttcacccca gttcatgtat ggcagccgcg cgttcacgaa gcaccccgca gatatccccg
     1201 attactataa acagtcattt cctgaggggt tcaagtggga acgagtaatg aactttgagg
     1261 atggtggcgc tgtaacggta acacaagata cctcgctcga agacggtacg ctcatttaca
     1321 aagtgaagct gcgcggaaca aatttccctc ctgatggacc agtaatgcag aaaaagacta
     1381 tgggatggga agcctcgacg gaacgtctct acccggagga cggggtgctg aaaggggata
     1441 ttaaaatggc actccgactg aaagacggcg gcaggtatct tgctgatttt aaaaccacgt
     1501 acaaagctaa aaagccagtg cagatgcctg gagcgtataa cgtagaccga aaactcgaca
     1561 ttacgtcgca taacgaggac tatacggtag tcgaacagta tgagaggtct gaagggaggc
     1621 attcaactgg tggtatggat gagttgtata agggatccgg atcaggatct ggatcaggat
     1681 cgggtagtta agagctccgc tgcgcttaaa tgtctttcat tataatcaat gtataacctt
     1741 tatgtagtat ttcaatctat gccgctgtgt acgtgcactg cggtgcctat ccttcggcat
     1801 tagagagtca ctgtttgtgt agatcgtagc tgcatgtctg acatctggtt cattagtgcc
     1861 gttttgagtc tgcactgttg tgcaacttga tattcatcac tgcacaccaa ccgtgtgttt
     1921 cctcctttgg gtgcaccctt taatccttgt cttctccttt tttgtctctt tccccctcga
     1981 aaaggtgtca aactactgcc gcataaacta cggttatccc aaatttaaga gaaagcaata
     2041 aagcatcagc tagcatgcct ttgtctcaag aagaatccac cctcattgaa agagcaacgg
     2101 ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca gctctctcta
     2161 gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga ccttgtgcag
     2221 aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact tgtatcgtcg
     2281 cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga caggtgcttc
     2341 tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag ccgacggcag
     2401 ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggctaactc gagtgcccat
     2461 ttagttggct tttcccttgt ctcgtgtctt ttccgtggaa aggttcccgg agtaatctga
     2521 tggcacagca gggaggtgcg cctgcaggtt ggttaggaag gggggatgat gtaaaagaag
     2581 aaaatggggg gattcgagcc ccgcacaggt ctctcaaatt ggatgcatag cttgagtatt
     2641 ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     2701 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     2761 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     2821 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2881 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2941 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3001 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3061 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3121 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3181 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3241 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3301 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3361 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3421 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3481 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3541 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3601 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3661 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3721 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3781 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3841 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3901 gcctgactcc ccgtcgtgta gataattacg atacgggagg gcttaccatc tggccccagt
     3961 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4021 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4081 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4141 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4201 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4261 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4321 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4381 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4441 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4501 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4561 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4621 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4681 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4741 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4801 cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag
     4861 gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     4921 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     4981 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     5041 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     5101 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     5161 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     5221 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     5281 acgctgcgcg taaccaccac acccgccgca cttaatgcgc cgctacaggg cgcgtccatt
     5341 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     5401 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     5461 cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca ctata
//
