LOCUS       sequence-495170-        6091 bp    DNA     circular SYN 11-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 6091)
  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 6091)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 11, 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-g418-3HA::FRB::3HA
FEATURES             Location/Qualifiers
     source          1..6091
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1009..1027
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1009..1026
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(1220..2014)
                     /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             complement(2411..2437)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2444..2470)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2477..2503)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2795..2821)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2828..2854)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2861..2887)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3278..4072)
                     /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"
     primer_bind     complement(4240..4259)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4241..4259)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4266..4283)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4266..4282)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4275..4297)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4423..4878
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4510..4529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4720..4741
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4956..5060
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5061..5129,5130..5921)
                     /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(5279..5298)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat ttaggtgaca
     1021 ctatagaata ctcaagctat gcatccaatt tgagagacct gtgcggggct cgaatccccc
     1081 cattttcttc ttttacatca tccccccttc ctaaccaacc tgcaggcgca cctccctgct
     1141 gtgccatcag attactccgg gaacctttcc acggaaaaga cacgagacaa gggaaaagcc
     1201 aactaaatgg gcactcgagt cagaagaact cgtcaagaag gcgatagaag gcgatgcgct
     1261 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa
     1321 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca
     1381 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc
     1441 aggcatcgcc gtgggtcacg acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg
     1501 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa
     1561 gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg
     1621 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt
     1681 tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca
     1741 gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg
     1801 tggccagcca cgatagccgc gctgcctcgt cctgcagttc attcagggca ccggacaggt
     1861 cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag
     1921 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg
     1981 gagaacctgc gtgcaatcca tcttgttcaa tcatgctagc tgatgcttta ttgctttctc
     2041 ttaaatttgg gataaccgta gtttatgcgg cagtagtttg acaccttttc gagggggaaa
     2101 gagacaaaaa aggagaagac aaggattaaa gggtgcaccc aaaggaggaa acacacggtt
     2161 ggtgtgcagt gatgaatatc aagttgcaca acagtgcaga ctcaaaacgg cactaatgaa
     2221 ccagatgtca gacatgcagc tacgatctac acaaacagtg actctctaat gccgaaggat
     2281 aggcaccgca gtgcacgtac acagcggcat agattgaaat actacataaa ggttatacat
     2341 tgattataat gaaagacatt taagcgcagc ggagctctta actacccgat cctgatccag
     2401 atcctgatcc cgcgtagtca gggacatcat acgggtaact gccggcgtag tccggtacat
     2461 catatggata tgatcccgca taatccggaa catcgtaggg gtaggatccc ttgctgattc
     2521 gacgaaacac atggtaatag aggtcccaag cctgcaggag atccttaacg ttcccagact
     2581 tcatgtattt tctacaccac tcttgtgctt ccattaggtc gcgcccgtaa gcctggttga
     2641 agctcgtctc cttcaacgtc tgtggaccac gctccatcat agcgtggagc ggctccaaca
     2701 cttcaaacat acccttcacg ttgcgctcac cgaaatatag tcgggacgct tcttccaatc
     2761 cctcgtgcca catctcgtgc cagagtataa gcttagcata atcgggtacg tcgtaaggat
     2821 acgaacctgc ataatctgga acgtcataag ggtacgaacc tgcgtaatct ggaacgtcgt
     2881 atgggtaaga accggaaccg gaaccactac cagaacccat aggtacccgt tgcagttgag
     2941 tttctgtttt cgggcttgtt ggtatacttg tttgattaat gtggctgata gctaagctat
     3001 cgcaaatatt tgtacaagac atgagggggc aaaaaaataa gccaagtgta tttgttaggc
     3061 ttccgtgcct gttagagaaa tatgtaagga aacgcctgcg ctacacagct tgagcaacgc
     3121 gtggggctcg aatcccccca ttttcttctt ttacatcatc cccccttcct aaccaacctg
     3181 caggcgcacc tccctgctgt gccatcagat tactccggga acctttccac ggaaaagaca
     3241 cgagacaagg gaaaggccaa ctaaatgggc accatggtca gaagaactcg tcaagaaggc
     3301 gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt
     3361 cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat
     3421 agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca
     3481 ccatgatatt cggcaagcag gcatcgccgt gggtcacgac gagatcctcg ccgtcgggca
     3541 tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca
     3601 gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt
     3661 tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat
     3721 cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg
     3781 gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg
     3841 cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat
     3901 tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc
     3961 ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc
     4021 tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgaattcca
     4081 ctagagctta ttttatggca gcaacgagac cttacgtaaa gcaaaactaa ataagagcgg
     4141 agactgcaat gcagagtaaa aaaaaaaaaa acatgaattt cagaagacct tgctgtgccc
     4201 ctagcttgca gcaggtctgc attatacggg cccaattcgc cctatagtga gtcgtattac
     4261 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4321 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4381 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg
     4441 cattaagtgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4501 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4561 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4621 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4681 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     4741 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     4801 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     4861 tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4921 acaccgcatc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4981 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5041 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5101 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5161 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5221 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5281 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5341 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5401 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5461 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5521 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5581 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5641 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5701 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5761 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5821 cccgtatcgt aattatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5881 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5941 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6001 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6061 cagaccccgt agaaaagatc aaaggatctt c
//
