LOCUS       sequence-374793-        5539 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 5539)
  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 5539)
  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-mGL
FEATURES             Location/Qualifiers
     source          1..5539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2664..2682)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2665..2682)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2700..2716)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2700..2716)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2713..2735)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2724..2740
                     /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(2748..2754,2755..2772,2773..2778))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2793..2814
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2931..2948)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3102..3690)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3182..3201)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3861..4652,4653..4721))
                     /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     4484..4503
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4722..4826)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4904..5359)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5041..5062)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5253..5272
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5485..5507
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5499..5516
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5500..5516
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5523..5539,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5523..5539,1..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 tctaagctta
      961 tggtatcaaa aggagaagag cttttcactg gggtcgtgcc tattttggtg gagttggatg
     1021 gggatgttaa tgggcataag ttctccgtgc gcggtgaagg cgaaggagac gctaccaacg
     1081 ggaagttaac gttgaaattt atatgtacga caggaaagtt gcccgtcccg tggccaacgc
     1141 ttgtcactac ccttggttac ggagtagcgt gcttcgcacg ttacccagat cacatgaaac
     1201 agcacgactt ctttaagtcc gctatgcccg aagggtacgt tcaagagaga acaatctcat
     1261 ttaaggacga tggaacgtac aagactagag cggaagttaa atttgagggg gatacacttg
     1321 ttaatcgtat agtcctgaag ggcatcgact ttaaggagga tggtaacata ctgggccaca
     1381 aactggagta caactttaat tcccacaaag tctacattac tgcggataaa cagaaaaacg
     1441 ggatcaaggc aaacttcaag acgcgccata acgtcgagga cggaggagtt caattggctg
     1501 accattacca acagaacacc ccaattggtg atggtcctgt actattgccg gacaatcatt
     1561 atctgagcca tcaaagtaaa ctatccaagg accccaatga aaaacgagat cacatggtac
     1621 ttaaagagcg ggtcacggcg gcggggatta cgcacgatat ggacgagttg tataagggat
     1681 ccggatcagg atctggatca ggatcgggta gttaagagct ccgctgcgct taaatgtctt
     1741 tcattataat caatgtataa cctttatgta gtatttcaat ctatgccgct gtgtacgtgc
     1801 actgcggtgc ctatccttcg gcattagaga gtcactgttt gtgtagatcg tagctgcatg
     1861 tctgacatct ggttcattag tgccgttttg agtctgcact gttgtgcaac ttgatattca
     1921 tcactgcaca ccaaccgtgt gtttcctcct ttgggtgcac cctttaatcc ttgtcttctc
     1981 cttttttgtc tctttccccc tcgaaaaggt gtcaaactac tgccgcataa actacggtta
     2041 tcccaaattt aagagaaagc aataaagcat cagctagcat gcctttgtct caagaagaat
     2101 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
     2161 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
     2221 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
     2281 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
     2341 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
     2401 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
     2461 gggagggcta actcgagtgc ccatttagtt ggcttttccc ttgtctcgtg tcttttccgt
     2521 ggaaaggttc ccggagtaat ctgatggcac agcagggagg tgcgcctgca ggttggttag
     2581 gaagggggga tgatgtaaaa gaagaaaatg gggggattcg agccccgcac aggtctctca
     2641 aattggatgc atagcttgag tattctatag tgtcacctaa atagcttggc gtaatcatgg
     2701 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     2761 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     2821 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2881 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     2941 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3001 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3061 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3121 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3181 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3241 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3301 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3361 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3421 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3481 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3541 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3601 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3661 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3721 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3781 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3841 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3901 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataat tacgatacgg
     3961 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4021 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4081 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4141 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4201 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4261 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4321 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4381 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4441 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4501 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4561 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4621 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4681 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4741 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4801 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgatgcggtg
     4861 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat
     4921 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     4981 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5041 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5101 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5161 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     5221 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     5281 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcacttaat
     5341 gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     5401 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     5461 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat
     5521 tgtaatacga ctcactata
//
