LOCUS       sequence-355571-        5915 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 5915)
  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 5915)
  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-3Ty:HaloTag:3Ty
FEATURES             Location/Qualifiers
     source          1..5915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             2452..2847
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AMPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(3040..3058)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3041..3058)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3076..3092)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3076..3092)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3089..3111)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3100..3116
                     /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(3124..3130,3131..3148,3149..3154))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3169..3190
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3307..3324)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3478..4066)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3558..3577)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4237..5028,5029..5097))
                     /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     4860..4879
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5098..5202)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5280..5735)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5417..5438)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5629..5648
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5861..5883
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5875..5892
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5876..5892
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5899..5915,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5899..5915,1..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 tctgaggtac
      961 acacaaacca ggacccactg gacggctccg aggtgcatac caatcaagat ccgctggacg
     1021 gctctgaagt tcacaccaat caagatccct tggataagct 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 gatccgaggt acatacaaat caggaccccc
     1981 tcgatggtag tgaagtccat acaaatcagg acccgctgga tggctcagag gttcatacga
     2041 accaagatcc tcttgatgga 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 agccatgcct
     2461 ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc
     2521 atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact
     2581 ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact
     2641 gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg
     2701 ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc
     2761 aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg
     2821 ccctctggtt atgtgtggga gggctaactc gagtgcccat ttagttggct tttcccttgt
     2881 ctcgtgtctt ttccgtggaa aggttcccgg agtaatctga tggcacagca gggaggtgcg
     2941 cctgcaggtt ggttaggaag gggggatgat gtaaaagaag aaaatggggg gattcgagcc
     3001 ccgcacaggt ctctcaaatt ggatgcatag cttgagtatt ctatagtgtc acctaaatag
     3061 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3121 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3181 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3241 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3301 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3361 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3421 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3481 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3541 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3601 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3661 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3721 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3781 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3841 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3901 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3961 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4021 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4081 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4141 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4201 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4261 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4321 gataattacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4381 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4441 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4501 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4561 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4621 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4681 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4741 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4801 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4861 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4921 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4981 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5041 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5101 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5161 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5221 gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga
     5281 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     5341 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     5401 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     5461 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     5521 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     5581 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     5641 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     5701 acccgccgca cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac
     5761 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     5821 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     5881 acgacggcca gtgaattgta atacgactca ctata
//
