LOCUS       sequence-374843-        5512 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 5512)
  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 5512)
  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-BFP
FEATURES             Location/Qualifiers
     source          1..5512
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2637..2655)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2638..2655)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2673..2689)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2673..2689)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2686..2708)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2697..2713
                     /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(2721..2727,2728..2745,2746..2751))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2766..2787
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2904..2921)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3075..3663)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3155..3174)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3834..4625,4626..4694))
                     /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     4457..4476
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4695..4799)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4877..5332)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5014..5035)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5226..5245
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5458..5480
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5472..5489
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5473..5489
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5496..5512,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5496..5512,1..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 gtgagttgat taaggaaaac atgcacatga agttgtacat ggaaggaaca gtagataatc
     1021 atcatttcaa atgtacgtca gaaggtgagg gcaaacctta tgaaggaact caaactatga
     1081 gaattaaagt cgttgagggt ggcccactac catttgcatt cgatatcctc gcaacatctt
     1141 ttctgtatgg ttctaaaacc tttatcaacc atacacaagg cataccagat ttctttaagc
     1201 aatcatttcc tgaaggtttc acatgggaaa gagtaactac ctatgaagat ggtggtgtcc
     1261 ttacagctac tcaagacacg tctttgcagg atggttgttt aatctacaat gttaagataa
     1321 gaggagtgaa ctttacatcc aatggaccag tcatgcaaaa gaaaactcta ggttgggagg
     1381 cattcacaga aactttgtac ccagccgatg ggggcttaga agggagaaat gacatggctt
     1441 taaaactggt tggcggtagc catttgattg cgaacgctaa aacaacctac agaagcaaaa
     1501 agccagctaa aaatcttaag atgcctgtat actacgtgga ttacagatta gaaaggaagg
     1561 aagctaataa tgaaacttac gttgaacagc acgaggtggc cgttgcccgt tattgcgacc
     1621 ttccttcaaa attgggacac aaactaaacg gatccggatc aggatctgga tcaggatcgg
     1681 gtagttaaga gctccgctgc gcttaaatgt ctttcattat aatcaatgta taacctttat
     1741 gtagtatttc aatctatgcc gctgtgtacg tgcactgcgg tgcctatcct tcggcattag
     1801 agagtcactg tttgtgtaga tcgtagctgc atgtctgaca tctggttcat tagtgccgtt
     1861 ttgagtctgc actgttgtgc aacttgatat tcatcactgc acaccaaccg tgtgtttcct
     1921 cctttgggtg caccctttaa tccttgtctt ctcctttttt gtctctttcc ccctcgaaaa
     1981 ggtgtcaaac tactgccgca taaactacgg ttatcccaaa tttaagagaa agcaataaag
     2041 catcagctag catgcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta
     2101 caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg
     2161 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac
     2221 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga
     2281 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg
     2341 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg
     2401 ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaactcgag tgcccattta
     2461 gttggctttt cccttgtctc gtgtcttttc cgtggaaagg ttcccggagt aatctgatgg
     2521 cacagcaggg aggtgcgcct gcaggttggt taggaagggg ggatgatgta aaagaagaaa
     2581 atggggggat tcgagccccg cacaggtctc tcaaattgga tgcatagctt gagtattcta
     2641 tagtgtcacc taaatagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     2701 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     2761 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2821 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2881 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2941 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3001 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3061 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3121 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3181 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3241 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3301 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3361 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3421 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3481 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     3541 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3601 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3661 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     3721 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3781 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     3841 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3901 tgactccccg tcgtgtagat aattacgata cgggagggct taccatctgg ccccagtgct
     3961 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4021 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4081 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4141 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4201 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     4261 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     4321 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4381 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     4441 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4501 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4561 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4621 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     4681 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4741 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     4801 acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag
     4861 aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     4921 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     4981 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     5041 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     5101 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     5161 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     5221 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     5281 ctgcgcgtaa ccaccacacc cgccgcactt aatgcgccgc tacagggcgc gtccattcgc
     5341 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     5401 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     5461 agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta ta
//
