LOCUS       sequence-374763-        5443 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 5443)
  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 5443)
  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-sfCherry2(1-10)
FEATURES             Location/Qualifiers
     source          1..5443
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2568..2586)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2569..2586)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2604..2620)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2604..2620)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2617..2639)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2628..2644
                     /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(2652..2658,2659..2676,2677..2682))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2697..2718
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2835..2852)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3006..3594)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3086..3105)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3765..4556,4557..4625))
                     /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     4388..4407
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4626..4730)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4808..5263)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4945..4966)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5157..5176
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5389..5411
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5403..5420
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5404..5420
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5427..5443,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5427..5443,1..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 aagaagataa catggccatt atcaaggaat ttatgcgctt caaagttcac atggaaggta
     1021 gtgtcaatgg tcacgagttc gagatagagg gagagggcga ggggcaccct tacgaaggta
     1081 cacagaccgc taaactcaaa gttaccaaag gtgggcctct tccctttgcc tgggatattc
     1141 ttagtccaca atttatgtat ggtagtaaag cctatgtgaa gcatccagct gatatccctg
     1201 attacctcaa actatcgttt ccagaaggct tcacctggga acgtgttatg aattttgagg
     1261 atggaggagt agtaacagtg acacaagact catcgttgca agacggacaa ttcatctaca
     1321 aagtgaaatt attaggcatt aactttccgt cggacggacc ggttatgcaa aaaaagacaa
     1381 tgggatggga agcgtctacg gaacggatgt accctgagga tggtgcactt aaaggtgaga
     1441 ttaatcagcg gttgaagctg aaggatggtg gacactatga cgccgaagtg aaaacgactt
     1501 acaaggcaaa aaaaccggtt cagcttccag gcgcgtataa cgtggacata aaactagata
     1561 tcacatcgca taacgaggat ggatccggat caggatctgg atcaggatcg ggtagttaag
     1621 agctccgctg cgcttaaatg tctttcatta taatcaatgt ataaccttta tgtagtattt
     1681 caatctatgc cgctgtgtac gtgcactgcg gtgcctatcc ttcggcatta gagagtcact
     1741 gtttgtgtag atcgtagctg catgtctgac atctggttca ttagtgccgt tttgagtctg
     1801 cactgttgtg caacttgata ttcatcactg cacaccaacc gtgtgtttcc tcctttgggt
     1861 gcacccttta atccttgtct tctccttttt tgtctctttc cccctcgaaa aggtgtcaaa
     1921 ctactgccgc ataaactacg gttatcccaa atttaagaga aagcaataaa gcatcagcta
     1981 gcatgccttt gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca
     2041 gcatccccat ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca
     2101 tcttcactgg tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc
     2161 tgggcactgc tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg
     2221 agaacagggg catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc
     2281 ctgggatcaa agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg
     2341 aattgctgcc ctctggttat gtgtgggagg gctaactcga gtgcccattt agttggcttt
     2401 tcccttgtct cgtgtctttt ccgtggaaag gttcccggag taatctgatg gcacagcagg
     2461 gaggtgcgcc tgcaggttgg ttaggaaggg gggatgatgt aaaagaagaa aatgggggga
     2521 ttcgagcccc gcacaggtct ctcaaattgg atgcatagct tgagtattct atagtgtcac
     2581 ctaaatagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2641 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     2701 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     2761 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2821 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2881 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2941 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3001 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3061 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3121 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3181 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3241 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3301 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3361 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3421 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3481 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3541 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3601 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3661 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3721 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3781 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3841 gtcgtgtaga taattacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3901 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3961 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4021 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4081 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4141 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4201 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4261 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4321 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4381 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4441 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4501 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4561 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4621 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4681 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4741 cgaaaagtgc cacctgatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     4801 catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     4861 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     4921 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     4981 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5041 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5101 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5161 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5221 accaccacac ccgccgcact taatgcgccg ctacagggcg cgtccattcg ccattcaggc
     5281 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     5341 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     5401 gttgtaaaac gacggccagt gaattgtaat acgactcact ata
//
