LOCUS       sequence-355324-        5791 bp    DNA     circular SYN 15-APR-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 5791)
  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 5791)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-birA*
FEATURES             Location/Qualifiers
     source          1..5791
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(963..1310,1311..1313,1314..1922)
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     promoter        complement(2916..2934)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2917..2934)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2952..2968)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2952..2968)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2965..2987)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2976..2992
                     /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(3000..3006,3007..3024,3025..3030))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3045..3066
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3183..3200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3354..3942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3434..3453)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4113..4904,4905..4973))
                     /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     4736..4755
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4974..5078)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5156..5611)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5293..5314)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5505..5524
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5737..5759
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5751..5768
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5752..5768
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5775..5791,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5775..5791,1..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 acaaggacaa caccgtgccc ctgaagctga tcgccctgct ggccaacggc gagttccact
     1021 ctggcgagca gctgggagag accctgggaa tgagcagagc cgccatcaac aagcacatcc
     1081 agacactgag agactgggga gtggacgtgt tcaccgtgcc tggcaagggc tacagcctgc
     1141 ctgagcctat ccagctgctg aacgccaagc agatcctggg acagctggat ggcggaagcg
     1201 tggccgtgct gcctgtgatc gactccacca atcagtacct gctggacaga atcggagagc
     1261 tgaagtccgg cgacgcctgc atcgccgagt accagcaggc tggcagagga ggcagaggac
     1321 ggaagtggtt cagcccattc ggagccaacc tgtacctgtc catgttctgg agactggagc
     1381 agggacctgc tgctgccatc ggactgagtc tggtgatcgg aatcgtgatg gccgaggtgc
     1441 tgagaaagct gggagccgac aaggtgagag tgaagtggcc taatgacctg tacctccagg
     1501 accgcaagct ggctggcatc ctggtggagc tgacaggcaa gacaggcgat gccgctcaga
     1561 tcgtgatcgg agccggaatc aacatggcca tgagaagagt ggaggagagc gtggtgaacc
     1621 agggctggat caccctgcag gaggctggca tcaacctgga ccggaacacc ctggccgcca
     1681 tgctgatcag agagctgaga gccgctctgg agctgttcga gcaggaggga ctggctcctt
     1741 acctgagcag atgggagaag ctggacaact tcatcaacag acctgtgaag ctgatcatcg
     1801 gcgacaagga aatcttcggc atctccagag gaatcgacaa gcagggagct ctgctgctgg
     1861 agcaggacgg aatcatcaag ccctggatgg gcggagaaat ctccctgaga agcgcagaga
     1921 agctcgaggg atccggatca ggatctggat caggatcggg tagttaagag ctccgctgcg
     1981 cttaaatgtc tttcattata atcaatgtat aacctttatg tagtatttca atctatgccg
     2041 ctgtgtacgt gcactgcggt gcctatcctt cggcattaga gagtcactgt ttgtgtagat
     2101 cgtagctgca tgtctgacat ctggttcatt agtgccgttt tgagtctgca ctgttgtgca
     2161 acttgatatt catcactgca caccaaccgt gtgtttcctc ctttgggtgc accctttaat
     2221 ccttgtcttc tccttttttg tctctttccc cctcgaaaag gtgtcaaact actgccgcat
     2281 aaactacggt tatcccaaat ttaagagaaa gcaataaagc atcagctagc atgcctttgt
     2341 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct
     2401 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg
     2461 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg
     2521 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca
     2581 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag
     2641 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct
     2701 ctggttatgt gtgggagggc taactcgagt gcccatttag ttggcttttc ccttgtctcg
     2761 tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga ggtgcgcctg
     2821 caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt cgagccccgc
     2881 acaggtctct caaattggat gcatagcttg agtattctat agtgtcacct aaatagcttg
     2941 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     3001 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     3061 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3121 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     3181 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3241 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3301 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3361 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3421 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3481 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3541 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3601 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3661 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3721 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3781 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3841 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3901 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3961 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4021 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     4081 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     4141 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4201 attacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     4261 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4321 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4381 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4441 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4501 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4561 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4621 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4681 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4741 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4801 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4861 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4921 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4981 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     5041 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     5101 cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt
     5161 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     5221 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5281 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5341 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     5401 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     5461 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     5521 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     5581 gccgcactta atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt
     5641 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     5701 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     5761 cggccagtga attgtaatac gactcactat a
//
