LOCUS       sequence-441439-        4915 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 4915)
  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 4915)
  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-blast-3HA
FEATURES             Location/Qualifiers
     source          1..4915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             875..901
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             908..934
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             941..967
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1955..1973)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1956..1973)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1991..2007)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1991..2007)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2004..2026)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2015..2031
                     /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(2039..2045,2046..2063,2064..2069))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2084..2105
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2222..2239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2393..2981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2473..2492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3152..3943,3944..4012))
                     /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     3775..3794
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4013..4117)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4195..4650)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4332..4353)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4544..4563
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4776..4798
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4790..4807
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4791..4807
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4814..4833
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4814..4832
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 actctgcatt gcagtctccg ctcttattta gttttgcttt acgtaaggtc tcgttgctgc
       61 cataaaataa gctctagtgg aattcatgcc tttgtctcaa gaagaatcca ccctcattga
      121 aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc
      181 agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg
      241 accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac
      301 ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg
      361 acaggtgctt ctcgacctgc atcctgggat caaagccata gtgaaggaca gtgatggaca
      421 gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaacc
      481 atggtgccca tttagttggc ctttcccttg tctcgtgtct tttccgtgga aaggttcccg
      541 gagtaatctg atggcacagc agggaggtgc gcctgcaggt tggttaggaa ggggggatga
      601 tgtaaaagaa gaaaatgggg ggattcgagc cccacgcgtt gctcaagctg tgtagcgcag
      661 gcgtttcctt acatatttct ctaacaggca cggaagccta acaaatacac ttggcttatt
      721 tttttgcccc ctcatgtctt gtacaaatat ttgcgatagc ttagctatca gccacattaa
      781 tcaaacaagt ataccaacaa gcccgaaaac agaaactcaa ctgcaacggg tacctatggg
      841 ttctggtagt ggttccggtt ccggttctaa gctttatccc tatgacgttc ctgactacgc
      901 tgggtcgtac ccttatgatg tgccggatta cgccggaagc tacccatacg atgtaccgga
      961 ctacgcagga tccggatcag gatctggatc aggatcgggt agttaagagc tccgctgcgc
     1021 ttaaatgtct ttcattataa tcaatgtata acctttatgt agtatttcaa tctatgccgc
     1081 tgtgtacgtg cactgcggtg cctatccttc ggcattagag agtcactgtt tgtgtagatc
     1141 gtagctgcat gtctgacatc tggttcatta gtgccgtttt gagtctgcac tgttgtgcaa
     1201 cttgatattc atcactgcac accaaccgtg tgtttcctcc tttgggtgca ccctttaatc
     1261 cttgtcttct ccttttttgt ctctttcccc ctcgaaaagg tgtcaaacta ctgccgcata
     1321 aactacggtt atcccaaatt taagagaaag caataaagca tcagctagca tgcctttgtc
     1381 tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc
     1441 tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt
     1501 caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc
     1561 tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat
     1621 cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc
     1681 catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc
     1741 tggttatgtg tgggagggct aactcgagtg cccatttagt tggcttttcc cttgtctcgt
     1801 gtcttttccg tggaaaggtt cccggagtaa tctgatggca cagcagggag gtgcgcctgc
     1861 aggttggtta ggaagggggg atgatgtaaa agaagaaaat ggggggattc gagccccgca
     1921 caggtctctc aaattggatg catagcttga gtattctata gtgtcaccta aatagcttgg
     1981 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     2041 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     2101 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     2161 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     2221 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2281 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2341 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2401 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2461 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2521 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2581 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2641 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2701 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2761 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2821 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2881 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2941 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3001 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3061 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3121 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3181 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3241 ttacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3301 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3361 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3421 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3481 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3541 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3601 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3661 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3721 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3781 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3841 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3901 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3961 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4021 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4081 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4141 ctgatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg
     4201 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     4261 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     4321 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     4381 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     4441 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     4501 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     4561 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     4621 ccgcacttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg
     4681 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     4741 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     4801 ggccagtgaa ttgtaatacg actcactata gggcgaattg ggcccgtata atgcagacct
     4861 gctgcaagct aggggcacag caaggtcttc tgaaattcat gttttttttt ttttt
//
