LOCUS       sequence-399937-        5756 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 5756)
  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 5756)
  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-3Ty:tagRFPt:3Ty
FEATURES             Location/Qualifiers
     source          1..5756
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2293..2688
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AMPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(2881..2899)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2882..2899)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2917..2933)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2917..2933)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2930..2952)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2941..2957
                     /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(2965..2971,2972..2989,2990..2995))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3010..3031
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3148..3165)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3319..3907)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3399..3418)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4078..4869,4870..4938))
                     /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     4701..4720
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4939..5043)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5121..5576)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5258..5279)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5470..5489
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5702..5724
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5716..5733
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5717..5733
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5740..5756,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5740..5756,1..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 atgcctttgt
      181 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct
      241 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg
      301 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg
      361 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca
      421 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga cctgcatcct gggatcaaag
      481 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct
      541 ctggttatgt gtgggagggc taaccatggt gcccatttag ttggcctttc ccttgtctcg
      601 tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga ggtgcgcctg
      661 caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt cgagccccac
      721 gcgttgctca agctgtgtag cgcaggcgtt tccttacata tttctctaac aggcacggaa
      781 gcctaacaaa tacacttggc ttattttttt gccccctcat gtcttgtaca aatatttgcg
      841 atagcttagc tatcagccac attaatcaaa caagtatacc aacaagcccg aaaacagaaa
      901 ctcaactgca acgggtacct atgggttctg gtagtggttc cggttccggt tctgaggtac
      961 acacaaacca ggacccactg gacggctccg aggtgcatac caatcaagat ccgctggacg
     1021 gctctgaagt tcacaccaat caagatccct tggataagct tgtctcgaag ggagaggagc
     1081 ttataaagga aaatatgcac atgaagttat atatggaagg aacggttaat aatcaccatt
     1141 ttaaatgtac gtcagaaggt gaaggaaagc catacgaggg cacacagact atgaggataa
     1201 aggttgtcga gggaggccca ctgcctttcg cgttcgatat tctcgccacg tcttttatgt
     1261 atggctcaag gactttcatc aatcatacgc agggcatccc ggactttttc aagcagagtt
     1321 ttcctgaggg gtttacgtgg gagcgtgtta ccacatacga agatggagga gtgcttaccg
     1381 caactcagga cacttcatta caagacggat gcctgatcta taacgttaag atccgcggag
     1441 tgaatttccc gagtaacgga ccagtgatgc aaaagaaaac acttggatgg gaagctaaca
     1501 cggagatgtt gtatcccgca gatgggggtt tggagggcag gacagacatg gcattaaaat
     1561 tggtgggcgg tggtcacctg atctgtaatt ttaagacgac gtatcgaagt aagaaacctg
     1621 ctaagaactt gaagatgcca ggggtctact acgtcgatca tcgccttgag aggattaaag
     1681 aggcagataa ggagacctat gtagaacagc acgaggtggc tgtggcccgt tactgcgatc
     1741 tgccctcgaa gttaggtcac aaattaaacg gcatggacga attgtacaag ggatccgagg
     1801 tacatacaaa tcaggacccc ctcgatggta gtgaagtcca tacaaatcag gacccgctgg
     1861 atggctcaga ggttcatacg aaccaagatc ctcttgatgg atcaggatct ggatcaggat
     1921 cgggtagtta agagctccgc tgcgcttaaa tgtctttcat tataatcaat gtataacctt
     1981 tatgtagtat ttcaatctat gccgctgtgt acgtgcactg cggtgcctat ccttcggcat
     2041 tagagagtca ctgtttgtgt agatcgtagc tgcatgtctg acatctggtt cattagtgcc
     2101 gttttgagtc tgcactgttg tgcaacttga tattcatcac tgcacaccaa ccgtgtgttt
     2161 cctcctttgg gtgcaccctt taatccttgt cttctccttt tttgtctctt tccccctcga
     2221 aaaggtgtca aactactgcc gcataaacta cggttatccc aaatttaaga gaaagcaata
     2281 aagcatcagc tagccatgcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
     2341 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
     2401 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
     2461 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
     2521 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
     2581 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
     2641 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaact cgagtgccca
     2701 tttagttggc ttttcccttg tctcgtgtct tttccgtgga aaggttcccg gagtaatctg
     2761 atggcacagc agggaggtgc gcctgcaggt tggttaggaa ggggggatga tgtaaaagaa
     2821 gaaaatgggg ggattcgagc cccgcacagg tctctcaaat tggatgcata gcttgagtat
     2881 tctatagtgt cacctaaata gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     2941 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3001 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3061 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3121 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3181 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3241 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3301 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3361 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3421 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3481 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3541 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3601 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3661 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3721 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3781 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3841 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3901 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3961 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4021 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4081 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4141 tgcctgactc cccgtcgtgt agataattac gatacgggag ggcttaccat ctggccccag
     4201 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4261 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4321 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4381 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4441 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4501 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4561 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4621 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4681 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4741 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4801 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4861 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4921 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4981 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5041 gcgcacattt ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa
     5101 ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5161 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5221 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5281 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5341 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5401 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5461 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     5521 cacgctgcgc gtaaccacca cacccgccgc acttaatgcg ccgctacagg gcgcgtccat
     5581 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     5641 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     5701 tcccagtcac gacgttgtaa aacgacggcc agtgaattgt aatacgactc actata
//
