LOCUS       sequence-355740-        7780 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 7780)
  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 7780)
  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-g418-3mNG
FEATURES             Location/Qualifiers
     source          1..7780
                     /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             3918..4712
                     /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(4905..4923)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4906..4923)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4941..4957)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4941..4957)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4954..4976)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4965..4981
                     /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(4989..4995,4996..5013,5014..5019))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5034..5055
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5172..5189)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5343..5931)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5423..5442)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6102..6893,6894..6962))
                     /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     6725..6744
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6963..7067)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7145..7600)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7282..7303)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7494..7513
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7726..7748
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7740..7757
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7741..7757
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(7764..7780,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(7764..7780,1..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 gttctaagct tgtctcgaaa ggtgaggaag
     1381 acaatatggc cagcttgcca gcaacgcacg agcttcatat atttgggtcg atcaacggtg
     1441 tggatttcga catggtgggt caaggcacag gtaatcctaa tgacgggtat gaagaactca
     1501 atttgaagag cactaaggga gatctccagt tttccccgtg gattcttgtg ccgcatattg
     1561 gttatggttt tcatcagtac ttaccatacc ccgatggaat gtcgcccttc caagcagcta
     1621 tggtggacgg ctcggggtat caagtacatc gcaccatgca atttgaggac ggagcatccc
     1681 tcaccgtaaa ctaccgctat acgtatgagg ggagccacat aaaaggtgag gctcaagtta
     1741 aaggcacagg cttcccagct gacggcccgg tcatgaccaa ttcattaacc gctgctgatt
     1801 ggtgccgttc taagaagacc taccctaacg acaagacgat cattagtact tttaagtgga
     1861 gttataccac aggaaacgga aagcggtatc gaagcactgc tcgaacaacc tacacatttg
     1921 cgaagccaat ggccgcgaac tacttgaaga accagccaat gtatgtgttt cgaaaaacgg
     1981 aactgaagca ttcgaaaacc gagcttaatt ttaaagagtg gcagaaggcg tttacagacg
     2041 tgatggggat ggacgaattg tataagcccg ggggcggctc cggaagcgtg tccaagggtg
     2101 aggaagataa catggcctct ctgcccgcca cccacgagtt gcatatcttt ggaagcatta
     2161 acggagtgga tttcgatatg gtgggtcagg ggacaggaaa tccgaacgat ggttatgaag
     2221 agttgaactt gaaaagcacg aagggagacc tccaattttc gccctggatc ttggtgccgc
     2281 atatcggata tgggttccat caatatttgc cctaccctga cgggatgtca ccctttcagg
     2341 ccgcgatggt tgatggttcg ggctatcagg tacaccggac aatgcaattt gaagatggtg
     2401 cctctttgac agtcaattac cggtacacct atgaagggag ccatattaaa ggtgaagccc
     2461 aagtgaaggg caccggcttc cctgcggacg ggcctgttat gacgaactca ctgactgcgg
     2521 cagattggtg taggtcgaag aagacttatc caaatgacaa aacaataatc tccacattta
     2581 agtggtcgta cacaacagga aatggtaagc gttaccgctc gactgcgagg acgacctaca
     2641 cattcgccaa gcctatggcg gccaactatc tcaagaatca acctatgtac gtgttccgga
     2701 aaactgagtt gaaacactct aaaaccgaac tcaacttcaa ggaatggcaa aaagcgttta
     2761 cggatgtaat gggaatggat gaattgtaca agggctccgg aagcggcagc gtatctaagg
     2821 gtgaagagga taatatggcg agcctgccag ccactcacga gcttcacatt ttcgggtcca
     2881 taaatggcgt agactttgac atggtaggac aggggacggg aaaccctaac gatggctatg
     2941 aggagcttaa cttgaaaagc acgaagggag atctgcaatt ctctccgtgg attctcgttc
     3001 cacacatagg ttatgggttt catcagtacc tcccctaccc agacggcatg agtccgttcc
     3061 aagcagcaat ggtcgatggt tctggctatc aagtccaccg tactatgcag tttgaagatg
     3121 gagcgagcct cacggtaaat tatcgttata cctacgaagg gtctcatatt aagggtgaag
     3181 cccaggtgaa ggggaccgga ttcccggccg atggccctgt gatgacgaat agccttacgg
     3241 ccgctgactg gtgtcggagt aaaaagacct atcccaatga caagacaatt atttccacgt
     3301 ttaaatggtc atatacgact gggaatggta agcgataccg atccactgcc cggacgactt
     3361 atacctttgc taagcctatg gcggcgaact acctcaagaa tcagccaatg tacgtgttcc
     3421 gaaagacaga gctgaaacac tcgaagactg aactcaattt taaagaatgg caaaaggcct
     3481 tcaccgatgt gatgggaatg gatgaactgt ataaaggatc cggatcagga tctggatcag
     3541 gatcgggtag ttaagagctc cgctgcgctt aaatgtcttt cattataatc aatgtataac
     3601 ctttatgtag tatttcaatc tatgccgctg tgtacgtgca ctgcggtgcc tatccttcgg
     3661 cattagagag tcactgtttg tgtagatcgt agctgcatgt ctgacatctg gttcattagt
     3721 gccgttttga gtctgcactg ttgtgcaact tgatattcat cactgcacac caaccgtgtg
     3781 tttcctcctt tgggtgcacc ctttaatcct tgtcttctcc ttttttgtct ctttccccct
     3841 cgaaaaggtg tcaaactact gccgcataaa ctacggttat cccaaattta agagaaagca
     3901 ataaagcatc agctagcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3961 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4021 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4081 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4141 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4201 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4261 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4321 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4381 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4441 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca cggcgatgcc tgcttgccga
     4501 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4561 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4621 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4681 ccttctatcg ccttcttgac gagttcttct gactcgagtg cccatttagt tggcttttcc
     4741 cttgtctcgt gtcttttccg tggaaaggtt cccggagtaa tctgatggca cagcagggag
     4801 gtgcgcctgc aggttggtta ggaagggggg atgatgtaaa agaagaaaat ggggggattc
     4861 gagccccgca caggtctctc aaattggatg catagcttga gtattctata gtgtcaccta
     4921 aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4981 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5041 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5101 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5161 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5221 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5281 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5341 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5401 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5461 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5521 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5581 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5641 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5701 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5761 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5821 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5881 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5941 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6001 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6061 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6121 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6181 gtgtagataa ttacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6241 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6301 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6361 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6421 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6481 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6541 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6601 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6661 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6721 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6781 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6841 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6901 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6961 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7021 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7081 aaagtgccac ctgatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     7141 caggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     7201 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     7261 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     7321 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     7381 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     7441 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     7501 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     7561 accacacccg ccgcacttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc
     7621 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     7681 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     7741 gtaaaacgac ggccagtgaa ttgtaatacg actcactata
//
