LOCUS       sequence-400064-        6367 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 6367)
  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 6367)
  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-g418-His:mNG:His
FEATURES             Location/Qualifiers
     source          1..6367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             171..965
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1356..1373
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2091..2108
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2505..3299
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(3492..3510)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3493..3510)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3528..3544)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3528..3544)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3541..3563)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3552..3568
                     /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(3576..3582,3583..3600,3601..3606))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3621..3642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3759..3776)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3930..4518)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4010..4029)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4689..5480,5481..5549))
                     /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     5312..5331
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5550..5654)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5732..6187)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5869..5890)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6081..6100
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6313..6335
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6327..6344
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6328..6344
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6351..6367,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6351..6367,1..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 atgattgaac
      181 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
      241 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
      301 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
      361 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
      421 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
      481 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
      541 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
      601 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
      661 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
      721 tcgtcgtgac ccacggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
      781 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
      841 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
      901 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
      961 tctgaccatg gtgcccattt agttggcctt tcccttgtct cgtgtctttt ccgtggaaag
     1021 gttcccggag taatctgatg gcacagcagg gaggtgcgcc tgcaggttgg ttaggaaggg
     1081 gggatgatgt aaaagaagaa aatgggggga ttcgagcccc acgcgttgct caagctgtgt
     1141 agcgcaggcg tttccttaca tatttctcta acaggcacgg aagcctaaca aatacacttg
     1201 gcttattttt ttgccccctc atgtcttgta caaatatttg cgatagctta gctatcagcc
     1261 acattaatca aacaagtata ccaacaagcc cgaaaacaga aactcaactg caacgggtac
     1321 ctatgggttc tggtagtggt tccggttccg gttctcacca ccaccaccat cacaagcttg
     1381 tctcgaaagg tgaggaagac aatatggcca gcttgccagc aacgcacgag cttcatatat
     1441 ttgggtcgat caacggtgtg gatttcgaca tggtgggtca aggcacaggt aatcctaatg
     1501 acgggtatga agaactcaat ttgaagagca ctaagggaga tctccagttt tccccgtgga
     1561 ttcttgtgcc gcatattggt tatggttttc atcagtactt accatacccc gatggaatgt
     1621 cgcccttcca agcagctatg gtggacggct cggggtatca agtacatcgc accatgcaat
     1681 ttgaggacgg agcatccctc accgtaaact accgctatac gtatgagggg agccacataa
     1741 aaggtgaggc tcaagttaaa ggcacaggct tcccagctga cggcccggtc atgaccaatt
     1801 cattaaccgc tgctgattgg tgccgttcta agaagaccta ccctaacgac aagacgatca
     1861 ttagtacttt taagtggagt tataccacag gaaacggaaa gcggtatcga agcactgctc
     1921 gaacaaccta cacatttgcg aagccaatgg ccgcgaacta cttgaagaac cagccaatgt
     1981 atgtgtttcg aaaaacggaa ctgaagcatt cgaaaaccga gcttaatttt aaagagtggc
     2041 agaaggcgtt tacagacgtg atggggatgg acgaattgta taagggatcc caccaccacc
     2101 accatcacgg atcaggatct ggatcaggat cgggtagtta agagctccgc tgcgcttaaa
     2161 tgtctttcat tataatcaat gtataacctt tatgtagtat ttcaatctat gccgctgtgt
     2221 acgtgcactg cggtgcctat ccttcggcat tagagagtca ctgtttgtgt agatcgtagc
     2281 tgcatgtctg acatctggtt cattagtgcc gttttgagtc tgcactgttg tgcaacttga
     2341 tattcatcac tgcacaccaa ccgtgtgttt cctcctttgg gtgcaccctt taatccttgt
     2401 cttctccttt tttgtctctt tccccctcga aaaggtgtca aactactgcc gcataaacta
     2461 cggttatccc aaatttaaga gaaagcaata aagcatcagc tagcatgatt gaacaagatg
     2521 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     2581 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     2641 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     2701 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     2761 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     2821 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     2881 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     2941 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     3001 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     3061 tgacccacgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3121 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     3181 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     3241 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgac
     3301 tcgagtgccc atttagttgg cttttccctt gtctcgtgtc ttttccgtgg aaaggttccc
     3361 ggagtaatct gatggcacag cagggaggtg cgcctgcagg ttggttagga aggggggatg
     3421 atgtaaaaga agaaaatggg gggattcgag ccccgcacag gtctctcaaa ttggatgcat
     3481 agcttgagta ttctatagtg tcacctaaat agcttggcgt aatcatggtc atagctgttt
     3541 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3601 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3661 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3721 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3781 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3841 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3901 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3961 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4021 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4081 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4141 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4201 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4261 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4321 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4381 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4441 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4501 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4561 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4621 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4681 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4741 tcatccatag ttgcctgact ccccgtcgtg tagataatta cgatacggga gggcttacca
     4801 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4861 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4921 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4981 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5041 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5101 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5161 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5221 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5281 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5341 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5401 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5461 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5521 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5581 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5641 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg atgcggtgtg aaataccgca
     5701 cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa
     5761 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     5821 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     5881 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     5941 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     6001 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     6061 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     6121 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cacttaatgc gccgctacag
     6181 ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     6241 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     6301 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact
     6361 cactata
//
