LOCUS       sequence-441448-        4924 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 4924)
  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 4924)
  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-3Ty
FEATURES             Location/Qualifiers
     source          1..4924
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(83..102)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(84..102)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(109..126)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(109..125)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(118..140)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      266..721
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(353..372)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     563..584
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        799..903
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(904..972,973..1764)
                     /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     complement(1122..1141)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1935..2523
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2424..2443
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2677..2694
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2811..2832
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2847..2852,2853..2870,2871..2877)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2885..2901
                     /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)."
     primer_bind     2890..2912
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2909..2925
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2909..2925
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2943..2961
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2943..2960
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 aaaaaaaaaa aaaacatgaa tttcagaaga ccttgctgtg cccctagctt gcagcaggtc
       61 tgcattatac gggcccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt
      121 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
      181 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
      241 gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag tgcggcgggt
      301 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
      361 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      421 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      481 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
      541 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
      601 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
      661 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
      721 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atcaggtggc
      781 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
      841 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
      901 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
      961 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     1021 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     1081 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     1141 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     1201 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     1261 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     1321 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     1381 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     1441 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     1501 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     1561 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     1621 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtaattatc
     1681 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     1741 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     1801 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     1861 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1921 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     1981 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2041 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     2101 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2161 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2221 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2281 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     2341 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     2401 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     2461 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     2521 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     2581 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     2641 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     2701 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     2761 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     2821 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     2881 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     2941 ctatttaggt gacactatag aatactcaag ctatgcatcc aatttgagag acctgtgcgg
     3001 ggctcgaatc cccccatttt cttcttttac atcatccccc cttcctaacc aacctgcagg
     3061 cgcacctccc tgctgtgcca tcagattact ccgggaacct ttccacggaa aagacacgag
     3121 acaagggaaa agccaactaa atgggcactc gagttagccc tcccacacat aaccagaggg
     3181 cagcaattca cgaatcccaa ctgccgtcgg ctgtccatca ctgtccttca ctatggcttt
     3241 gatcccagga tgcagatcga gaagcacctg tcggcaccgt ccgcaggggc tcaagatgcc
     3301 cctgttctca tttccgatcg cgacgataca agtcaggttg ccagctgccg cagcagcagc
     3361 agtgcccagc accacgagtt ctgcacaagg tcccccagta aaatgatata cattgacacc
     3421 agtgaagatg cggccgtcgc tagagagagc tgcgctggcg acgctgtagt cttcagagat
     3481 ggggatgctg ttgattgtag ccgttgctct ttcaatgagg gtggattctt cttgagacaa
     3541 aggcatgcta gctgatgctt tattgctttc tcttaaattt gggataaccg tagtttatgc
     3601 ggcagtagtt tgacaccttt tcgaggggga aagagacaaa aaaggagaag acaaggatta
     3661 aagggtgcac ccaaaggagg aaacacacgg ttggtgtgca gtgatgaata tcaagttgca
     3721 caacagtgca gactcaaaac ggcactaatg aaccagatgt cagacatgca gctacgatct
     3781 acacaaacag tgactctcta atgccgaagg ataggcaccg cagtgcacgt acacagcggc
     3841 atagattgaa atactacata aaggttatac attgattata atgaaagaca tttaagcgca
     3901 gcggagctct taactacccg atcctgatcc agatcctgat ccggatccgt ccagagggtc
     3961 ctgattagtg tgcacttcgg agccatccaa gggatcttga ttggtgtgaa cttcagagcc
     4021 gtccagcgga tcttgattgg tatgcacctc aagcttagaa ccggaaccgg aaccactacc
     4081 agaacccata ggtacccgtt gcagttgagt ttctgttttc gggcttgttg gtatacttgt
     4141 ttgattaatg tggctgatag ctaagctatc gcaaatattt gtacaagaca tgagggggca
     4201 aaaaaataag ccaagtgtat ttgttaggct tccgtgcctg ttagagaaat atgtaaggaa
     4261 acgcctgcgc tacacagctt gagcaacgcg tggggctcga atccccccat tttcttcttt
     4321 tacatcatcc ccccttccta accaacctgc aggcgcacct ccctgctgtg ccatcagatt
     4381 actccgggaa cctttccacg gaaaagacac gagacaaggg aaaggccaac taaatgggca
     4441 ccatggttag ccctcccaca cataaccaga gggcagcaat tcacgaatcc caactgccgt
     4501 cggctgtcca tcactgtcct tcactatggc tttgatccca ggatgcaggt cgagaagcac
     4561 ctgtcggcac cgtccgcagg ggctcaagat gcccctgttc tcatttccga tcgcgacgat
     4621 acaagtcagg ttgccagctg ccgcagcagc agcagtgccc agcaccacga gttctgcaca
     4681 aggtccccca gtaaaatgat atacattgac accagtgaag atgcggccgt cgctagagag
     4741 agctgcgctg gcgacgctgt agtcttcaga gatggggatg ctgttgattg tagccgttgc
     4801 tctttcaatg agggtggatt cttcttgaga caaaggcatg aattccacta gagcttattt
     4861 tatggcagca acgagacctt acgtaaagca aaactaaata agagcggaga ctgcaatgca
     4921 gagt
//
