LOCUS       sequence-355748-        5732 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 5732)
  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 5732)
  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:mNG:3Ty
FEATURES             Location/Qualifiers
     source          1..5732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2269..2664
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AMPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(2857..2875)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2858..2875)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2893..2909)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2893..2909)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2906..2928)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2917..2933
                     /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(2941..2947,2948..2965,2966..2971))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2986..3007
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3124..3141)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3295..3883)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3375..3394)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4054..4845,4846..4914))
                     /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     4677..4696
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4915..5019)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5097..5552)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5234..5255)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5446..5465
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5678..5700
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5692..5709
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5693..5709
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5716..5732,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5716..5732,1..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 tgtctcgaaa ggtgaggaag
     1081 acaatatggc cagcttgcca gcaacgcacg agcttcatat atttgggtcg atcaacggtg
     1141 tggatttcga catggtgggt caaggcacag gtaatcctaa tgacgggtat gaagaactca
     1201 atttgaagag cactaaggga gatctccagt tttccccgtg gattcttgtg ccgcatattg
     1261 gttatggttt tcatcagtac ttaccatacc ccgatggaat gtcgcccttc caagcagcta
     1321 tggtggacgg ctcggggtat caagtacatc gcaccatgca atttgaggac ggagcatccc
     1381 tcaccgtaaa ctaccgctat acgtatgagg ggagccacat aaaaggtgag gctcaagtta
     1441 aaggcacagg cttcccagct gacggcccgg tcatgaccaa ttcattaacc gctgctgatt
     1501 ggtgccgttc taagaagacc taccctaacg acaagacgat cattagtact tttaagtgga
     1561 gttataccac aggaaacgga aagcggtatc gaagcactgc tcgaacaacc tacacatttg
     1621 cgaagccaat ggccgcgaac tacttgaaga accagccaat gtatgtgttt cgaaaaacgg
     1681 aactgaagca ttcgaaaacc gagcttaatt ttaaagagtg gcagaaggcg tttacagacg
     1741 tgatggggat ggacgaattg tataagggat ccgaggtaca tacaaatcag gaccccctcg
     1801 atggtagtga agtccataca aatcaggacc cgctggatgg ctcagaggtt catacgaacc
     1861 aagatcctct tgatggatca ggatctggat caggatcggg tagttaagag ctccgctgcg
     1921 cttaaatgtc tttcattata atcaatgtat aacctttatg tagtatttca atctatgccg
     1981 ctgtgtacgt gcactgcggt gcctatcctt cggcattaga gagtcactgt ttgtgtagat
     2041 cgtagctgca tgtctgacat ctggttcatt agtgccgttt tgagtctgca ctgttgtgca
     2101 acttgatatt catcactgca caccaaccgt gtgtttcctc ctttgggtgc accctttaat
     2161 ccttgtcttc tccttttttg tctctttccc cctcgaaaag gtgtcaaact actgccgcat
     2221 aaactacggt tatcccaaat ttaagagaaa gcaataaagc atcagctagc catgcctttg
     2281 tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc
     2341 tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat cttcactggt
     2401 gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct
     2461 gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc
     2521 atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa
     2581 gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc
     2641 tctggttatg tgtgggaggg ctaactcgag tgcccattta gttggctttt cccttgtctc
     2701 gtgtcttttc cgtggaaagg ttcccggagt aatctgatgg cacagcaggg aggtgcgcct
     2761 gcaggttggt taggaagggg ggatgatgta aaagaagaaa atggggggat tcgagccccg
     2821 cacaggtctc tcaaattgga tgcatagctt gagtattcta tagtgtcacc taaatagctt
     2881 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     2941 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3001 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3061 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3121 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3181 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3241 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3301 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3361 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3421 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3481 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3541 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3601 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3661 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3721 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3781 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3841 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3901 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3961 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4021 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4081 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4141 aattacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4201 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4261 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4321 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4381 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4441 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4501 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4561 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4621 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4681 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4741 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4801 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4861 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4921 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4981 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5041 acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat
     5101 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     5161 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     5221 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     5281 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     5341 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     5401 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     5461 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     5521 cgccgcactt aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt
     5581 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     5641 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     5701 acggccagtg aattgtaata cgactcacta ta
//
