LOCUS       sequence-355745-        5533 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 5533)
  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 5533)
  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-sfGFP
FEATURES             Location/Qualifiers
     source          1..5533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             960..1670
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="RKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     promoter        complement(2658..2676)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2659..2676)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2694..2710)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2694..2710)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2707..2729)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2718..2734
                     /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(2742..2748,2749..2766,2767..2772))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2787..2808
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2925..2942)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3096..3684)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3176..3195)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3855..4646,4647..4715))
                     /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     4478..4497
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4716..4820)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4898..5353)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5035..5056)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5247..5266
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5479..5501
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5493..5510
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5494..5510
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5517..5533,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5517..5533,1..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 tctaagctta
      961 ggaagggaga ggagttgttc actggtgtgg tgccgatcct cgtcgaactt gacggagatg
     1021 ttaatggaca taaattctct gtccgagggg aaggcgaagg tgatgcaact aacgggaaac
     1081 tcacgttgaa attcatatgt actactggta agctgcccgt tccgtggcca actctcgtga
     1141 cgacattaac ctatggcgtg cagtgttttg ccaggtaccc ggatcatatg aagcagcacg
     1201 actttttcaa aagcgcgatg ccggagggct atgttcagga gcgcacgatc agctttaaag
     1261 atgatggcac ctacaagact cgggccgagg tcaagtttga aggtgatact ttagtaaatc
     1321 gcatcgaatt gaaaggcata gacttcaaag aagatggcaa cattttaggc cacaagctgg
     1381 agtacaattt caattcccac aatgtgtata taaccgcaga caaacagaag aatggtatta
     1441 aagcgaattt taagatacgt cacaatgtcg aagacgggag tgtgcaactc gcggaccact
     1501 accagcagaa cacacccata ggtgacggcc ccgttttgct tccggacaac cattacctgt
     1561 ctactcagtc ggtattgtcc aaggacccga acgaaaaacg ggaccatatg gtgttgctgg
     1621 agttcgttac agcagcaggt ataacgcacg gaatggatga gctttacaag ggatccggat
     1681 caggatctgg atcaggatcg ggtagttaag agctccgctg cgcttaaatg tctttcatta
     1741 taatcaatgt ataaccttta tgtagtattt caatctatgc cgctgtgtac gtgcactgcg
     1801 gtgcctatcc ttcggcatta gagagtcact gtttgtgtag atcgtagctg catgtctgac
     1861 atctggttca ttagtgccgt tttgagtctg cactgttgtg caacttgata ttcatcactg
     1921 cacaccaacc gtgtgtttcc tcctttgggt gcacccttta atccttgtct tctccttttt
     1981 tgtctctttc cccctcgaaa aggtgtcaaa ctactgccgc ataaactacg gttatcccaa
     2041 atttaagaga aagcaataaa gcatcagcta gcatgccttt gtctcaagaa gaatccaccc
     2101 tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg
     2161 ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta tatcatttta
     2221 ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca
     2281 acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggac
     2341 ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg
     2401 atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg
     2461 gctaactcga gtgcccattt agttggcttt tcccttgtct cgtgtctttt ccgtggaaag
     2521 gttcccggag taatctgatg gcacagcagg gaggtgcgcc tgcaggttgg ttaggaaggg
     2581 gggatgatgt aaaagaagaa aatgggggga ttcgagcccc gcacaggtct ctcaaattgg
     2641 atgcatagct tgagtattct atagtgtcac ctaaatagct tggcgtaatc atggtcatag
     2701 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     2761 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     2821 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     2881 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     2941 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3001 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3061 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3121 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3181 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3241 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3301 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3361 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3421 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3481 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     3541 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     3601 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     3661 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     3721 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     3781 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3841 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3901 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taattacgat acgggagggc
     3961 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4021 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4081 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4141 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4201 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4261 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4321 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4381 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4441 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4501 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4561 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     4621 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     4681 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     4741 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4801 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat
     4861 accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taatattttg
     4921 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4981 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     5041 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     5101 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     5161 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     5221 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     5281 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcact taatgcgccg
     5341 ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     5401 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     5461 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat
     5521 acgactcact ata
//
