LOCUS       sequence-355079-        6247 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 6247)
  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 6247)
  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: pPOTv7-blast-tdTomato
FEATURES             Location/Qualifiers
     source          1..6247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             960..2384
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWGRVMNFEDGGL
                     VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
                     ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
                     LFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYE
                     GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMN
                     FEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVL
                     KGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYER
                     SEGRHHLFLYGMDELYK"
     promoter        complement(3372..3390)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3373..3390)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3408..3424)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3408..3424)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3421..3443)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3432..3448
                     /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(3456..3462,3463..3480,3481..3486))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3501..3522
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3639..3656)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3810..4398)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3890..3909)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4569..5360,5361..5429))
                     /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     5192..5211
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5430..5534)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5612..6067)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5749..5770)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5961..5980
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6193..6215
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6207..6224
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6208..6224
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6231..6247,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6231..6247,1..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 tctaagcttg
      961 tgagcaaggg cgaggaggtc atcaaagagt tcatgcgctt caaggtgcgc atggagggct
     1021 ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1081 cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc
     1141 tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg
     1201 attacaagaa gctgtccttc cccgagggct tcaagtgggg gcgcgtgatg aacttcgagg
     1261 acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg ctgatctaca
     1321 aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag aagaagacca
     1381 tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga
     1441 tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc aagaccatct
     1501 acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc aagctggaca
     1561 tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc gagggccgcc
     1621 accacctgtt cctggggcat ggcaccggca gcaccggcag cggcagctcc ggcaccgcct
     1681 cctccgagga caacaacatg gccgtcatca aagagttcat gcgcttcaag gtgcgcatgg
     1741 agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1801 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
     1861 acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca
     1921 tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     1981 tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga
     2041 tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga
     2101 agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg
     2161 gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga
     2221 ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc
     2281 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg
     2341 gccgccacca cctgttcctg tacggcatgg acgagctgta caagggatcc ggatcaggat
     2401 ctggatcagg atcgggtagt taagagctcc gctgcgctta aatgtctttc attataatca
     2461 atgtataacc tttatgtagt atttcaatct atgccgctgt gtacgtgcac tgcggtgcct
     2521 atccttcggc attagagagt cactgtttgt gtagatcgta gctgcatgtc tgacatctgg
     2581 ttcattagtg ccgttttgag tctgcactgt tgtgcaactt gatattcatc actgcacacc
     2641 aaccgtgtgt ttcctccttt gggtgcaccc tttaatcctt gtcttctcct tttttgtctc
     2701 tttccccctc gaaaaggtgt caaactactg ccgcataaac tacggttatc ccaaatttaa
     2761 gagaaagcaa taaagcatca gctagcatgc ctttgtctca agaagaatcc accctcattg
     2821 aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg
     2881 cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg
     2941 gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga
     3001 cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc
     3061 gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac
     3121 agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaac
     3181 tcgagtgccc atttagttgg cttttccctt gtctcgtgtc ttttccgtgg aaaggttccc
     3241 ggagtaatct gatggcacag cagggaggtg cgcctgcagg ttggttagga aggggggatg
     3301 atgtaaaaga agaaaatggg gggattcgag ccccgcacag gtctctcaaa ttggatgcat
     3361 agcttgagta ttctatagtg tcacctaaat agcttggcgt aatcatggtc atagctgttt
     3421 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3481 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3541 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3601 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3661 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3721 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3781 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3841 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3901 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3961 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4021 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4081 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4141 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4201 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4261 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4321 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4381 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4441 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4501 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4561 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4621 tcatccatag ttgcctgact ccccgtcgtg tagataatta cgatacggga gggcttacca
     4681 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4741 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4801 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4861 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4921 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4981 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5041 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5101 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5161 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5221 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5281 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5341 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5401 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5461 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5521 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg atgcggtgtg aaataccgca
     5581 cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa
     5641 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     5701 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     5761 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     5821 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     5881 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     5941 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     6001 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cacttaatgc gccgctacag
     6061 ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     6121 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     6181 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact
     6241 cactata
//
