LOCUS       sequence-400013-        5723 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 5723)
  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 5723)
  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:mScarletI:3Ty
FEATURES             Location/Qualifiers
     source          1..5723
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2260..2655
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AMPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(2848..2866)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2849..2866)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2884..2900)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2884..2900)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2897..2919)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2908..2924
                     /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(2932..2938,2939..2956,2957..2962))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2977..2998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3115..3132)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3286..3874)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3366..3385)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4045..4836,4837..4905))
                     /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     4668..4687
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4906..5010)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5088..5543)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5225..5246)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5437..5456
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5669..5691
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5683..5700
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5684..5700
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5707..5723,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5707..5723,1..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 tatggtatca aagggcgaag
     1081 cagtaatcaa ggagttcatg cggtttaagg tacacatgga gggttccatg aatggccatg
     1141 aatttgagat cgaaggagag ggggaagggc gtccttacga aggaacacaa acagctaaac
     1201 ttaaggtaac taagggggga cccctcccct tttcctggga catactgtca ccacaattca
     1261 tgtacggcag ccgagccttt ataaaacatc cagctgatat acccgattat tataagcaaa
     1321 gttttcccga gggctttaag tgggaaaggg tcatgaactt cgaggacggg ggtgcggtaa
     1381 ctgtaaccca agacacctct ctcgaagacg ggacgcttat ttataaggtg aaactccggg
     1441 ggaccaattt tccccctgac ggacctgtca tgcagaagaa gacaatgggc tgggaggcgt
     1501 ccacagagcg cctgtatccg gaggatggcg tacttaaggg tgacataaaa atggcacttc
     1561 gtttgaagga cggtggccgt tatctcgctg actttaagac aacttataag gcgaaaaagc
     1621 cggtacagat gccaggagca tacaatgttg accgcaaatt ggacatcacc agccacaacg
     1681 aagactatac cgtcgttgaa cagtacgaac ggagtgaagg gaggcacagt acaggcggga
     1741 tggacgaatt gtataaagga tccgaggtac atacaaatca ggaccccctc gatggtagtg
     1801 aagtccatac aaatcaggac ccgctggatg gctcagaggt tcatacgaac caagatcctc
     1861 ttgatggatc aggatctgga tcaggatcgg gtagttaaga gctccgctgc gcttaaatgt
     1921 ctttcattat aatcaatgta taacctttat gtagtatttc aatctatgcc gctgtgtacg
     1981 tgcactgcgg tgcctatcct tcggcattag agagtcactg tttgtgtaga tcgtagctgc
     2041 atgtctgaca tctggttcat tagtgccgtt ttgagtctgc actgttgtgc aacttgatat
     2101 tcatcactgc acaccaaccg tgtgtttcct cctttgggtg caccctttaa tccttgtctt
     2161 ctcctttttt gtctctttcc ccctcgaaaa ggtgtcaaac tactgccgca taaactacgg
     2221 ttatcccaaa tttaagagaa agcaataaag catcagctag ccatgccttt gtctcaagaa
     2281 gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac
     2341 tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta
     2401 tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg
     2461 gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc
     2521 ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg
     2581 aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat
     2641 gtgtgggagg gctaactcga gtgcccattt agttggcttt tcccttgtct cgtgtctttt
     2701 ccgtggaaag gttcccggag taatctgatg gcacagcagg gaggtgcgcc tgcaggttgg
     2761 ttaggaaggg gggatgatgt aaaagaagaa aatgggggga ttcgagcccc gcacaggtct
     2821 ctcaaattgg atgcatagct tgagtattct atagtgtcac ctaaatagct tggcgtaatc
     2881 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2941 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3001 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3061 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3121 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3181 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3241 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3301 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3361 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3421 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3481 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3541 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3601 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3661 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3721 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3781 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3841 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3901 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3961 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4021 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4081 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taattacgat
     4141 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4201 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4261 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4321 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4381 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4441 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4501 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4561 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4621 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4681 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4741 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4801 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4861 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4921 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4981 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgatgc
     5041 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt
     5101 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     5161 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     5221 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     5281 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     5341 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     5401 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     5461 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcact
     5521 taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg
     5581 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     5641 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     5701 gaattgtaat acgactcact ata
//
