LOCUS       sequence-355147-        5551 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 5551)
  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 5551)
  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-tagRFPt
FEATURES             Location/Qualifiers
     source          1..5551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2676..2694)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2677..2694)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2712..2728)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2712..2728)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2725..2747)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2736..2752
                     /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(2760..2766,2767..2784,2785..2790))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2805..2826
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2943..2960)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3114..3702)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3194..3213)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3873..4664,4665..4733))
                     /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     4496..4515
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4734..4838)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4916..5371)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5053..5074)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5265..5284
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5497..5519
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5511..5528
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5512..5528
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5535..5551,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5535..5551,1..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 tctcgaaggg agaggagctt ataaaggaaa atatgcacat gaagttatat atggaaggaa
     1021 cggttaataa tcaccatttt aaatgtacgt cagaaggtga aggaaagcca tacgagggca
     1081 cacagactat gaggataaag gttgtcgagg gaggcccact gcctttcgcg ttcgatattc
     1141 tcgccacgtc ttttatgtat ggctcaagga ctttcatcaa tcatacgcag ggcatcccgg
     1201 actttttcaa gcagagtttt cctgaggggt ttacgtggga gcgtgttacc acatacgaag
     1261 atggaggagt gcttaccgca actcaggaca cttcattaca agacggatgc ctgatctata
     1321 acgttaagat ccgcggagtg aatttcccga gtaacggacc agtgatgcaa aagaaaacac
     1381 ttggatggga agctaacacg gagatgttgt atcccgcaga tgggggtttg gagggcagga
     1441 cagacatggc attaaaattg gtgggcggtg gtcacctgat ctgtaatttt aagacgacgt
     1501 atcgaagtaa gaaacctgct aagaacttga agatgccagg ggtctactac gtcgatcatc
     1561 gccttgagag gattaaagag gcagataagg agacctatgt agaacagcac gaggtggctg
     1621 tggcccgtta ctgcgatctg ccctcgaagt taggtcacaa attaaacggc atggacgaat
     1681 tgtacaaggg atccggatca ggatctggat caggatcggg tagttaagag ctccgctgcg
     1741 cttaaatgtc tttcattata atcaatgtat aacctttatg tagtatttca atctatgccg
     1801 ctgtgtacgt gcactgcggt gcctatcctt cggcattaga gagtcactgt ttgtgtagat
     1861 cgtagctgca tgtctgacat ctggttcatt agtgccgttt tgagtctgca ctgttgtgca
     1921 acttgatatt catcactgca caccaaccgt gtgtttcctc ctttgggtgc accctttaat
     1981 ccttgtcttc tccttttttg tctctttccc cctcgaaaag gtgtcaaact actgccgcat
     2041 aaactacggt tatcccaaat ttaagagaaa gcaataaagc atcagctagc atgcctttgt
     2101 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct
     2161 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg
     2221 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg
     2281 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca
     2341 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag
     2401 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct
     2461 ctggttatgt gtgggagggc taactcgagt gcccatttag ttggcttttc ccttgtctcg
     2521 tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga ggtgcgcctg
     2581 caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt cgagccccgc
     2641 acaggtctct caaattggat gcatagcttg agtattctat agtgtcacct aaatagcttg
     2701 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2761 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2821 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2881 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2941 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3001 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3061 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3121 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3181 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3241 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3301 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3361 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3421 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3481 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3541 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3601 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3661 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3721 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3781 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3841 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3901 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3961 attacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     4021 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4081 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4141 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4201 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4261 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4321 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4381 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4441 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4501 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4561 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4621 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4681 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4741 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4801 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4861 cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt
     4921 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     4981 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5041 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5101 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     5161 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     5221 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     5281 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     5341 gccgcactta atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt
     5401 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     5461 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     5521 cggccagtga attgtaatac gactcactat a
//
