LOCUS       sequence-440821-        5562 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 5562)
  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 5562)
  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-His:mNG:His
FEATURES             Location/Qualifiers
     source          1..5562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             953..970
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1688..1705
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(2687..2705)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2688..2705)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2723..2739)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2723..2739)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2736..2758)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2747..2763
                     /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(2771..2777,2778..2795,2796..2801))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2816..2837
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2954..2971)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3125..3713)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3205..3224)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3884..4675,4676..4744))
                     /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     4507..4526
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4745..4849)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4927..5382)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5064..5085)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5276..5295
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5508..5530
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5522..5539
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5523..5539
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5546..5562,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5546..5562,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgtata atgcagacct gctgcaagct aggggcacag caaggtcttc
       61 tgaaattcat gttttttttt ttttactctg cattgcagtc tccgctctta tttagttttg
      121 ctttacgtaa ggtctcgttg ctgccataaa ataagctcta gtggaattca tgcctttgtc
      181 tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc
      241 tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt
      301 caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc
      361 tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat
      421 cttgagcccc tgcggacggt gccgacaggt gcttctcgac ctgcatcctg ggatcaaagc
      481 catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc
      541 tggttatgtg tgggagggct aaccatggtg cccatttagt tggcctttcc cttgtctcgt
      601 gtcttttccg tggaaaggtt cccggagtaa tctgatggca cagcagggag gtgcgcctgc
      661 aggttggtta ggaagggggg atgatgtaaa agaagaaaat ggggggattc gagccccacg
      721 cgttgctcaa gctgtgtagc gcaggcgttt ccttacatat ttctctaaca ggcacggaag
      781 cctaacaaat acacttggct tatttttttg ccccctcatg tcttgtacaa atatttgcga
      841 tagcttagct atcagccaca ttaatcaaac aagtatacca acaagcccga aaacagaaac
      901 tcaactgcaa cgggtaccta tgggttctgg tagtggttcc ggttccggtt ctcaccacca
      961 ccaccatcac aagcttgtct cgaaaggtga ggaagacaat atggccagct tgccagcaac
     1021 gcacgagctt catatatttg ggtcgatcaa cggtgtggat ttcgacatgg tgggtcaagg
     1081 cacaggtaat cctaatgacg ggtatgaaga actcaatttg aagagcacta agggagatct
     1141 ccagttttcc ccgtggattc ttgtgccgca tattggttat ggttttcatc agtacttacc
     1201 ataccccgat ggaatgtcgc ccttccaagc agctatggtg gacggctcgg ggtatcaagt
     1261 acatcgcacc atgcaatttg aggacggagc atccctcacc gtaaactacc gctatacgta
     1321 tgaggggagc cacataaaag gtgaggctca agttaaaggc acaggcttcc cagctgacgg
     1381 cccggtcatg accaattcat taaccgctgc tgattggtgc cgttctaaga agacctaccc
     1441 taacgacaag acgatcatta gtacttttaa gtggagttat accacaggaa acggaaagcg
     1501 gtatcgaagc actgctcgaa caacctacac atttgcgaag ccaatggccg cgaactactt
     1561 gaagaaccag ccaatgtatg tgtttcgaaa aacggaactg aagcattcga aaaccgagct
     1621 taattttaaa gagtggcaga aggcgtttac agacgtgatg gggatggacg aattgtataa
     1681 gggatcccac caccaccacc atcacggatc aggatctgga tcaggatcgg gtagttaaga
     1741 gctccgctgc gcttaaatgt ctttcattat aatcaatgta taacctttat gtagtatttc
     1801 aatctatgcc gctgtgtacg tgcactgcgg tgcctatcct tcggcattag agagtcactg
     1861 tttgtgtaga tcgtagctgc atgtctgaca tctggttcat tagtgccgtt ttgagtctgc
     1921 actgttgtgc aacttgatat tcatcactgc acaccaaccg tgtgtttcct cctttgggtg
     1981 caccctttaa tccttgtctt ctcctttttt gtctctttcc ccctcgaaaa ggtgtcaaac
     2041 tactgccgca taaactacgg ttatcccaaa tttaagagaa agcaataaag catcagctag
     2101 catgcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag
     2161 catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat
     2221 cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct
     2281 gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga
     2341 gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc
     2401 tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga
     2461 attgctgccc tctggttatg tgtgggaggg ctaactcgag tgcccattta gttggctttt
     2521 cccttgtctc gtgtcttttc cgtggaaagg ttcccggagt aatctgatgg cacagcaggg
     2581 aggtgcgcct gcaggttggt taggaagggg ggatgatgta aaagaagaaa atggggggat
     2641 tcgagccccg cacaggtctc tcaaattgga tgcatagctt gagtattcta tagtgtcacc
     2701 taaatagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     2761 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     2821 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     2881 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2941 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3001 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3061 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3121 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3181 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3241 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3301 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3361 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3421 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3481 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3541 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3601 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3661 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3721 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3781 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3841 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3901 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3961 tcgtgtagat aattacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     4021 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4081 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4141 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4201 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4261 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4321 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4381 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4441 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4501 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4561 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4621 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4681 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4741 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4801 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4861 gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     4921 atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     4981 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     5041 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     5101 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     5161 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     5221 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     5281 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     5341 ccaccacacc cgccgcactt aatgcgccgc tacagggcgc gtccattcgc cattcaggct
     5401 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     5461 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     5521 ttgtaaaacg acggccagtg aattgtaata cgactcacta ta
//
