LOCUS       sequence-495165-        6769 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 6769)
  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 6769)
  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-g418-3HA::tbTurboID::3HA
FEATURES             Location/Qualifiers
     source          1..6769
                     /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(3473..3499)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3506..3532)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3539..3565)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3956..4750)
                     /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(4918..4937)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4919..4937)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4944..4961)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4944..4960)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4953..4975)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5101..5556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5188..5207)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5398..5419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5634..5738
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5739..5807,5808..6599)
                     /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(5957..5976)
                     /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 ttctcggcag
     2521 acctaaggga gatctcgccg cccatccaag gtttgataac accgtcttgt tccaacaaca
     2581 atgctccctg tttgtctatg cctcgtgaaa tgccaaaaat ttctttatct ccaataataa
     2641 gtttcaccgg tcggttgata aagttatcta acttttccca gcggggaagg tagggtgcga
     2701 ggccttcctg ttcgaataac tccaaggcgg cccgaagttc cctaatgagg gttgccgcca
     2761 gcgtattccg atccaggttt attccggctt cctgcagggt tatccaaccc tgattgacca
     2821 cgctctcctc gaccctgcgc atcgccacgt taatacctgc accaatcacg atttgtgcgg
     2881 catcgcccgt tataccggct aattcgacaa gaatacccgc caatttacga tcttgtaaat
     2941 acagatcgtt gggccatttg accctgactt tatctgcccc aagtttgcgt agtgcttccg
     3001 ccattacaat gccaattact ggacctagac ctattgccgc cggacctctc ttaagtctcc
     3061 agaacatgct cagataaagg ttcgccccga atggactaaa ccactttcga ccccgggacc
     3121 cgcgtccggc ctgctgatat tcagcaatgc aagcatcacc tgacttgagt tcacctatgc
     3181 gatctaacag gtattggtta gtggaatcaa ccacggggag gacagccaca ctgccgccat
     3241 cgagctgccc taggatctgc tttgcgttta aaagaggaat tggctctggt agcgaatacc
     3301 cttttccagg cacagtaaac acatccactc cccaatcacg taacgtctga atgtgcttat
     3361 taatagcagc acgcgacatg ccgagggttt ctcccaactg ttcaccactg tgaaattccc
     3421 cattagcaag gagcgcgata agttttagtg gcaccgtgtt atctttaagc ttagcataat
     3481 cgggtacgtc gtaaggatac gaacctgcat aatctggaac gtcataaggg tacgaacctg
     3541 cgtaatctgg aacgtcgtat gggtaagaac cggaaccgga accactacca gaacccatag
     3601 gtacccgttg cagttgagtt tctgttttcg ggcttgttgg tatacttgtt tgattaatgt
     3661 ggctgatagc taagctatcg caaatatttg tacaagacat gagggggcaa aaaaataagc
     3721 caagtgtatt tgttaggctt ccgtgcctgt tagagaaata tgtaaggaaa cgcctgcgct
     3781 acacagcttg agcaacgcgt ggggctcgaa tccccccatt ttcttctttt acatcatccc
     3841 cccttcctaa ccaacctgca ggcgcacctc cctgctgtgc catcagatta ctccgggaac
     3901 ctttccacgg aaaagacacg agacaaggga aaggccaact aaatgggcac catggtcaga
     3961 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
     4021 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
     4081 ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag
     4141 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccgtgg gtcacgacga
     4201 gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc
     4261 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg
     4321 ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat
     4381 gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg
     4441 acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga
     4501 caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg
     4561 cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc
     4621 gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc
     4681 agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt
     4741 gttcaatcat gaattccact agagcttatt ttatggcagc aacgagacct tacgtaaagc
     4801 aaaactaaat aagagcggag actgcaatgc agagtaaaaa aaaaaaaaac atgaatttca
     4861 gaagaccttg ctgtgcccct agcttgcagc aggtctgcat tatacgggcc caattcgccc
     4921 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     4981 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     5041 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     5101 acgcgccctg tagcggcgca ttaagtgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5161 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5221 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5281 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5341 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5401 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5461 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5521 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
     5581 gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat gtgcgcggaa
     5641 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5701 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5761 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5821 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5881 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5941 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6001 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6061 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6121 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6181 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6241 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6301 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6361 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6421 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6481 ggccagatgg taagccctcc cgtatcgtaa ttatctacac gacggggagt caggcaacta
     6541 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6601 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6661 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6721 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
