LOCUS       sequence-495167-        5286 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 5286)
  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 5286)
  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-3HA::FRB::3HA
FEATURES             Location/Qualifiers
     source          1..5286
                     /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(2009..2035)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2042..2068)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2075..2101)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2393..2419)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2426..2452)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2459..2485)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3435..3454)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3436..3454)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3461..3478)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3461..3477)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3470..3492)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3618..4073
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3705..3724)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3915..3936
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4151..4255
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4256..4324,4325..5116)
                     /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(4474..4493)
                     /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 tagccctccc acacataacc agagggcagc aattcacgaa
     1261 tcccaactgc cgtcggctgt ccatcactgt ccttcactat ggctttgatc ccaggatgca
     1321 gatcgagaag cacctgtcgg caccgtccgc aggggctcaa gatgcccctg ttctcatttc
     1381 cgatcgcgac gatacaagtc aggttgccag ctgccgcagc agcagcagtg cccagcacca
     1441 cgagttctgc acaaggtccc ccagtaaaat gatatacatt gacaccagtg aagatgcggc
     1501 cgtcgctaga gagagctgcg ctggcgacgc tgtagtcttc agagatgggg atgctgttga
     1561 ttgtagccgt tgctctttca atgagggtgg attcttcttg agacaaaggc atgctagctg
     1621 atgctttatt gctttctctt aaatttggga taaccgtagt ttatgcggca gtagtttgac
     1681 accttttcga gggggaaaga gacaaaaaag gagaagacaa ggattaaagg gtgcacccaa
     1741 aggaggaaac acacggttgg tgtgcagtga tgaatatcaa gttgcacaac agtgcagact
     1801 caaaacggca ctaatgaacc agatgtcaga catgcagcta cgatctacac aaacagtgac
     1861 tctctaatgc cgaaggatag gcaccgcagt gcacgtacac agcggcatag attgaaatac
     1921 tacataaagg ttatacattg attataatga aagacattta agcgcagcgg agctcttaac
     1981 tacccgatcc tgatccagat cctgatcccg cgtagtcagg gacatcatac gggtaactgc
     2041 cggcgtagtc cggtacatca tatggatatg atcccgcata atccggaaca tcgtaggggt
     2101 aggatccctt gctgattcga cgaaacacat ggtaatagag gtcccaagcc tgcaggagat
     2161 ccttaacgtt cccagacttc atgtattttc tacaccactc ttgtgcttcc attaggtcgc
     2221 gcccgtaagc ctggttgaag ctcgtctcct tcaacgtctg tggaccacgc tccatcatag
     2281 cgtggagcgg ctccaacact tcaaacatac ccttcacgtt gcgctcaccg aaatatagtc
     2341 gggacgcttc ttccaatccc tcgtgccaca tctcgtgcca gagtataagc ttagcataat
     2401 cgggtacgtc gtaaggatac gaacctgcat aatctggaac gtcataaggg tacgaacctg
     2461 cgtaatctgg aacgtcgtat gggtaagaac cggaaccgga accactacca gaacccatag
     2521 gtacccgttg cagttgagtt tctgttttcg ggcttgttgg tatacttgtt tgattaatgt
     2581 ggctgatagc taagctatcg caaatatttg tacaagacat gagggggcaa aaaaataagc
     2641 caagtgtatt tgttaggctt ccgtgcctgt tagagaaata tgtaaggaaa cgcctgcgct
     2701 acacagcttg agcaacgcgt ggggctcgaa tccccccatt ttcttctttt acatcatccc
     2761 cccttcctaa ccaacctgca ggcgcacctc cctgctgtgc catcagatta ctccgggaac
     2821 ctttccacgg aaaagacacg agacaaggga aaggccaact aaatgggcac catggttagc
     2881 cctcccacac ataaccagag ggcagcaatt cacgaatccc aactgccgtc ggctgtccat
     2941 cactgtcctt cactatggct ttgatcccag gatgcaggtc gagaagcacc tgtcggcacc
     3001 gtccgcaggg gctcaagatg cccctgttct catttccgat cgcgacgata caagtcaggt
     3061 tgccagctgc cgcagcagca gcagtgccca gcaccacgag ttctgcacaa ggtcccccag
     3121 taaaatgata tacattgaca ccagtgaaga tgcggccgtc gctagagaga gctgcgctgg
     3181 cgacgctgta gtcttcagag atggggatgc tgttgattgt agccgttgct ctttcaatga
     3241 gggtggattc ttcttgagac aaaggcatga attccactag agcttatttt atggcagcaa
     3301 cgagacctta cgtaaagcaa aactaaataa gagcggagac tgcaatgcag agtaaaaaaa
     3361 aaaaaacatg aatttcagaa gaccttgctg tgcccctagc ttgcagcagg tctgcattat
     3421 acgggcccaa ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac
     3481 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3541 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     3601 gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agtgcggcgg gtgtggtggt
     3661 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3721 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3781 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3841 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3901 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3961 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4021 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg
     4081 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatcaggtg gcacttttcg
     4141 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4201 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     4261 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4321 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     4381 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4441 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4501 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4561 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4621 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4681 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4741 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     4801 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4861 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4921 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4981 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtaatta tctacacgac
     5041 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5101 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5161 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5221 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5281 atcttc
//
