LOCUS       sequence-355568-        5914 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 5914)
  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 5914)
  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: pPOTv6-blast-3Myc:HaloTag:3Myc
FEATURES             Location/Qualifiers
     source          1..5914
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             954..983
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             990..1019
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1026..1055
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1062..1949
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     CDS             1956..1985
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1992..2021
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2028..2057
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     promoter        complement(3039..3057)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3040..3057)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3075..3091)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3075..3091)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3088..3110)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3099..3115
                     /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(3123..3129,3130..3147,3148..3153))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3168..3189
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3306..3323)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3477..4065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3557..3576)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4236..5027,5028..5096))
                     /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     4859..4878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5097..5201)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5279..5734)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5416..5437)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5628..5647
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5860..5882
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5874..5891
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5875..5891
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5898..5914,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5898..5914,1..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 tctgagcaga
      961 agctgataag tgaagaagac ttgggctccg agcaaaagct gatttcggaa gaggatctgg
     1021 gctctgagca gaagctgatt tctgaagagg atcttaagct tgctgagatt ggtacggggt
     1081 ttccttttga cccgcactac gttgaggtgt tgggtgaacg tatgcattat gttgatgttg
     1141 gtccccgtga tggtacgccc gtgctgtttt tgcatggtaa ccccacgtca agctatgtgt
     1201 ggaggaacat tattccccac gtggcaccta ctcatcgctg tattgctcca gaccttattg
     1261 gtatgggtaa gtcagataaa ccggatcttg gttacttctt tgacgatcac gtgcgtttta
     1321 tggatgcatt tattgaagcg cttggccttg aagaagttgt gttagtgatt cacgattggg
     1381 gttctgcctt gggatttcat tgggctaaac gtaatcccga aagagttaaa ggtattgcat
     1441 tcatggagtt cattcgtcct attcctacgt gggatgagtg gccagagttt gcgcgcgaga
     1501 cgtttcaagc gtttcgtaca acggacgtgg gtcgtaagct gattattgat caaaatgttt
     1561 ttattgaggg tacgctgccg atgggtgttg tgcgtcccct gactgaagtg gagatggatc
     1621 actaccgtga gccattttta aatccagtgg accgtgagcc attgtggcgc ttccctaacg
     1681 agctgcccat tgctggtgaa ccggctaaca ttgtggcgct tgtggaggag tatatggatt
     1741 ggcttcatca atcacccgtg cctaagttgc ttttttgggg tacgccaggt gtgcttattc
     1801 cacctgctga agcagcacgc ttggctaaat cacttccaaa ctgcaaagca gttgatattg
     1861 gtccgggtct gaacctgttg caagaagata atcccgatct tattggttcc gagattgccc
     1921 gttggctttc gacacttgaa atttctggtg gatccgagca gaagctgata agcgaggaag
     1981 acctgggtag tgaacagaag ctgatctcag aagaggacct tggctcagaa caaaaactca
     2041 tctcagagga agatcttgga tcaggatctg gatcaggatc gggtagttaa gagctccgct
     2101 gcgcttaaat gtctttcatt ataatcaatg tataaccttt atgtagtatt tcaatctatg
     2161 ccgctgtgta cgtgcactgc ggtgcctatc cttcggcatt agagagtcac tgtttgtgta
     2221 gatcgtagct gcatgtctga catctggttc attagtgccg ttttgagtct gcactgttgt
     2281 gcaacttgat attcatcact gcacaccaac cgtgtgtttc ctcctttggg tgcacccttt
     2341 aatccttgtc ttctcctttt ttgtctcttt ccccctcgaa aaggtgtcaa actactgccg
     2401 cataaactac ggttatccca aatttaagag aaagcaataa agcatcagct agcatgcctt
     2461 tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca
     2521 tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg
     2581 gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg
     2641 ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg
     2701 gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca
     2761 aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc
     2821 cctctggtta tgtgtgggag ggctaactcg agtgcccatt tagttggctt ttcccttgtc
     2881 tcgtgtcttt tccgtggaaa ggttcccgga gtaatctgat ggcacagcag ggaggtgcgc
     2941 ctgcaggttg gttaggaagg ggggatgatg taaaagaaga aaatgggggg attcgagccc
     3001 cgcacaggtc tctcaaattg gatgcatagc ttgagtattc tatagtgtca cctaaatagc
     3061 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3121 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3181 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3241 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3301 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3361 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3421 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3481 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3541 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3601 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3661 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3721 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3781 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3841 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3901 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3961 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4021 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4081 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4141 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4201 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4261 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4321 ataattacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4381 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4441 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4501 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4561 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4621 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4681 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4741 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4801 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4861 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4921 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4981 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5041 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5101 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5161 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5221 ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa
     5281 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     5341 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     5401 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     5461 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     5521 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     5581 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     5641 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     5701 cccgccgcac ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact
     5761 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     5821 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     5881 cgacggccag tgaattgtaa tacgactcac tata
//
