LOCUS       sequence-356273-        5546 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 5546)
  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 5546)
  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: pPOTv6.1-phleo-Ty:mNG:Ty
FEATURES             Location/Qualifiers
     source          1..5546
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2671..2689)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2672..2689)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2707..2723)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2707..2723)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2720..2742)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2731..2747
                     /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(2755..2761,2762..2779,2780..2785))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2800..2821
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2938..2955)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3109..3697)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3189..3208)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3868..4659,4660..4728))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4491..4510
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4729..4833)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4911..5366)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5048..5069)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5260..5279
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5492..5514
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5506..5523
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5507..5523
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5530..5546,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5530..5546,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgtata atgcagacct gctgcaagct aggggcacag caaggtcttc
       61 tgaaattcat gttttttttt tttttactct gcattgcagt ctccgctctt atttagtttt
      121 gctttacgta aggtctcgtt gctgccataa aataagctct agtggaattc atggctaaac
      181 tcacttctgc tgtaccagta ctcactgcta gagatgtagc tggagctgta gagttctgga
      241 ctgatagact cggattctct agagatttcg tagaggatga tttcgctgga gtagtaagag
      301 atgatgtaac tctcttcata tctgctgtac aagatcaagt agtaccagat aacactctcg
      361 cttgggtatg ggtaagagga ctcgatgagc tctacgctga gtggtctgag gtagtatcta
      421 ctaacttcag agatgcttct ggaccagcta tgactgagat aggagagcaa ccgtggggaa
      481 gagagttcgc tctcagagat ccagctggaa actgcgtaca cttcgtagct gaggagcaag
      541 attaaccatg gtgcccattt agttggcttt tcccttgtct cgtgtctttt ccgtggaaag
      601 gttcccggag taatctgatg gcacagcagg gaggtgcgcc tgcaggttgg ttaggaaggg
      661 gggatgatgt aaaagaagaa aatgggggga ttcgagcccc acgcgttgct caagctgtgt
      721 agcgcaggcg tttccttaca tatttctcta acaggcacgg aagcctaaca aatacacttg
      781 gcttattttt ttgccccctc atgtcttgta caaatatttg cgatagctta gctatcagcc
      841 acattaatca aacaagtata ccaacaagcc cgaaaacaga aactcaactg caacgggtac
      901 ctatgggttc tggtagtggt tccggttccg gttctgaggt acacacaaac caggacccac
      961 tggacggctc cgaggtgcat accaatcaag atccgctgga cggctctgaa gttcacacca
     1021 atcaagatcc cttggataag cttgtctcga aaggtgagga agacaatatg gccagcttgc
     1081 cagcaacgca cgagcttcat atatttgggt cgatcaacgg tgtggatttc gacatggtgg
     1141 gtcaaggcac aggtaatcct aatgacgggt atgaagaact caatttgaag agcactaagg
     1201 gagatctcca gttttccccg tggattcttg tgccgcatat tggttatggt tttcatcagt
     1261 acttaccata ccccgatgga atgtcgccct tccaagcagc tatggtggac ggctcggggt
     1321 atcaagtaca tcgcaccatg caatttgagg acggagcatc cctcaccgta aactaccgct
     1381 atacgtatga ggggagccac ataaaaggtg aggctcaagt taaaggcaca ggcttcccag
     1441 ctgacggccc ggtcatgacc aattcattaa ccgctgctga ttggtgccgt tctaagaaga
     1501 cctaccctaa cgacaagacg atcattagta cttttaagtg gagttatacc acaggaaacg
     1561 gaaagcggta tcgaagcact gctcgaacaa cctacacatt tgcgaagcca atggccgcga
     1621 actacttgaa gaaccagcca atgtatgtgt ttcgaaaaac ggaactgaag cattcgaaaa
     1681 ccgagcttaa ttttaaagag tggcagaagg cgtttacaga cgtgatgggg atggacgaat
     1741 tgtataaggg atccgaggta catacaaatc aggaccccct cgatggtagt gaagtccata
     1801 caaatcagga cccgctggat ggctcagagg ttcatacgaa ccaagatcct cttgatggat
     1861 caggatctgg atcaggatcg ggtagttaag agctccgctg cgcttaaatg tctttcatta
     1921 taatcaatgt ataaccttta tgtagtattt caatctatgc cgctgtgtac gtgcactgcg
     1981 gtgcctatcc ttcggcatta gagagtcact gtttgtgtag atcgtagctg catgtctgac
     2041 atctggttca ttagtgccgt tttgagtctg cactgttgtg caacttgata ttcatcactg
     2101 cacaccaacc gtgtgtttcc tcctttgggt gcacccttta atccttgtct tctccttttt
     2161 tgtctctttc cccctcgaaa aggtgtcaaa ctactgccgc ataaactacg gttatcccaa
     2221 atttaagaga aagcaataaa gcatcagcta gcatggctaa actcacttct gctgtaccag
     2281 tactcactgc tagagatgta gctggagctg tagagttctg gactgataga ctcggattct
     2341 ctagagattt cgtagaggat gatttcgctg gagtagtaag agatgatgta actctcttca
     2401 tatctgctgt acaagatcaa gtagtaccag ataacactct cgcttgggta tgggtaagag
     2461 gactcgatga gctctacgct gagtggtctg aggtagtatc tactaacttc agagatgctt
     2521 ctggaccagc tatgactgag ataggagagc aaccgtgggg aagagagttc gctctcagag
     2581 atccagctgg aaactgcgta cacttcgtag ctgaggagca agattaactc gaggcacagg
     2641 tctctcaaat tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta
     2701 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2761 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2821 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2881 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2941 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3001 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3061 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3121 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3181 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3241 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3301 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3361 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3421 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3481 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3541 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3601 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3661 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3721 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3781 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3841 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3901 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3961 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4021 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4081 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4141 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4201 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4261 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4321 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4381 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4441 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4501 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4561 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4621 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4681 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4741 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4801 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     4861 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag
     4921 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     4981 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5041 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5101 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5161 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5221 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     5281 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     5341 gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa
     5401 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     5461 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     5521 agtgaattgt aatacgactc actata
//
