LOCUS       sequence-495188-        5964 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 5964)
  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 5964)
  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-blast-3HA::tbTurboID::3HA
FEATURES             Location/Qualifiers
     source          1..5964
                     /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(3071..3097)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3104..3130)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3137..3163)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4113..4132)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4114..4132)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4139..4156)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4139..4155)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4148..4170)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4296..4751
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4383..4402)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4593..4614
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4829..4933
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4934..5002,5003..5794)
                     /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(5152..5171)
                     /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 ctcggcagac ctaagggaga tctcgccgcc catccaaggt ttgataacac
     2161 cgtcttgttc caacaacaat gctccctgtt tgtctatgcc tcgtgaaatg ccaaaaattt
     2221 ctttatctcc aataataagt ttcaccggtc ggttgataaa gttatctaac ttttcccagc
     2281 ggggaaggta gggtgcgagg ccttcctgtt cgaataactc caaggcggcc cgaagttccc
     2341 taatgagggt tgccgccagc gtattccgat ccaggtttat tccggcttcc tgcagggtta
     2401 tccaaccctg attgaccacg ctctcctcga ccctgcgcat cgccacgtta atacctgcac
     2461 caatcacgat ttgtgcggca tcgcccgtta taccggctaa ttcgacaaga atacccgcca
     2521 atttacgatc ttgtaaatac agatcgttgg gccatttgac cctgacttta tctgccccaa
     2581 gtttgcgtag tgcttccgcc attacaatgc caattactgg acctagacct attgccgccg
     2641 gacctctctt aagtctccag aacatgctca gataaaggtt cgccccgaat ggactaaacc
     2701 actttcgacc ccgggacccg cgtccggcct gctgatattc agcaatgcaa gcatcacctg
     2761 acttgagttc acctatgcga tctaacaggt attggttagt ggaatcaacc acggggagga
     2821 cagccacact gccgccatcg agctgcccta ggatctgctt tgcgtttaaa agaggaattg
     2881 gctctggtag cgaataccct tttccaggca cagtaaacac atccactccc caatcacgta
     2941 acgtctgaat gtgcttatta atagcagcac gcgacatgcc gagggtttct cccaactgtt
     3001 caccactgtg aaattcccca ttagcaagga gcgcgataag ttttagtggc accgtgttat
     3061 ctttaagctt agcataatcg ggtacgtcgt aaggatacga acctgcataa tctggaacgt
     3121 cataagggta cgaacctgcg taatctggaa cgtcgtatgg gtaagaaccg gaaccggaac
     3181 cactaccaga acccataggt acccgttgca gttgagtttc tgttttcggg cttgttggta
     3241 tacttgtttg attaatgtgg ctgatagcta agctatcgca aatatttgta caagacatga
     3301 gggggcaaaa aaataagcca agtgtatttg ttaggcttcc gtgcctgtta gagaaatatg
     3361 taaggaaacg cctgcgctac acagcttgag caacgcgtgg ggctcgaatc cccccatttt
     3421 cttcttttac atcatccccc cttcctaacc aacctgcagg cgcacctccc tgctgtgcca
     3481 tcagattact ccgggaacct ttccacggaa aagacacgag acaagggaaa ggccaactaa
     3541 atgggcacca tggttagccc tcccacacat aaccagaggg cagcaattca cgaatcccaa
     3601 ctgccgtcgg ctgtccatca ctgtccttca ctatggcttt gatcccagga tgcaggtcga
     3661 gaagcacctg tcggcaccgt ccgcaggggc tcaagatgcc cctgttctca tttccgatcg
     3721 cgacgataca agtcaggttg ccagctgccg cagcagcagc agtgcccagc accacgagtt
     3781 ctgcacaagg tcccccagta aaatgatata cattgacacc agtgaagatg cggccgtcgc
     3841 tagagagagc tgcgctggcg acgctgtagt cttcagagat ggggatgctg ttgattgtag
     3901 ccgttgctct ttcaatgagg gtggattctt cttgagacaa aggcatgaat tccactagag
     3961 cttattttat ggcagcaacg agaccttacg taaagcaaaa ctaaataaga gcggagactg
     4021 caatgcagag taaaaaaaaa aaaacatgaa tttcagaaga ccttgctgtg cccctagctt
     4081 gcagcaggtc tgcattatac gggcccaatt cgccctatag tgagtcgtat tacaattcac
     4141 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     4201 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     4261 cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag
     4321 tgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     4381 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     4441 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4501 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     4561 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     4621 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     4681 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     4741 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4801 atcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4861 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     4921 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     4981 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5041 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5101 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5161 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5221 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5281 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5341 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5401 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5461 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5521 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5581 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5641 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5701 cgtaattatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5761 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5821 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5881 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5941 cgtagaaaag atcaaaggat cttc
//
