LOCUS       sequence-441435-        5470 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 5470)
  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 5470)
  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-mStayGold[E138D]
FEATURES             Location/Qualifiers
     source          1..5470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(81..100)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(82..100)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(107..124)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(107..123)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(116..138)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      264..719
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(351..370)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     561..582
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        797..901
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(902..970,971..1762)
                     /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     complement(1120..1139)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1933..2521
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2422..2441
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2675..2692
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2809..2830
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2845..2850,2851..2868,2869..2875)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2883..2899
                     /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)."
     primer_bind     2888..2910
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2907..2923
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2907..2923
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2941..2959
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2941..2958
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(3947..4594)
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ivorra-Molla et al., 2023)"
                     /label=mStayGold (E138D)
                     /note="contains the monomerizing E138D mutation"
                     /translation="ASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKLP
                     MSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFMK
                     NGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNDVQHIPRDDGVECPVTLLYPLLSDKSK
                     CVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL"
ORIGIN
        1 aaaaaaaaaa aacatgaatt tcagaagacc ttgctgtgcc cctagcttgc agcaggtctg
       61 cattatacgg gcccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt
      121 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
      181 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
      241 tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagtg cggcgggtgt
      301 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
      361 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
      421 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
      481 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
      541 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
      601 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
      661 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc
      721 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat caggtggcac
      781 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
      841 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
      901 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
      961 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     1021 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     1081 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     1141 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     1201 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     1261 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     1321 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     1381 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     1441 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     1501 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     1561 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     1621 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg taattatcta
     1681 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     1741 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     1801 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     1861 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     1921 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1981 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2041 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2101 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2161 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2221 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2281 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2341 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2401 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2461 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     2521 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     2581 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     2641 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     2701 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     2761 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     2821 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     2881 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     2941 atttaggtga cactatagaa tactcaagct atgcatccaa tttgagagac ctgtgcgggg
     3001 ctcgaatccc cccattttct tcttttacat catcccccct tcctaaccaa cctgcaggcg
     3061 cacctccctg ctgtgccatc agattactcc gggaaccttt ccacggaaaa gacacgagac
     3121 aagggaaaag ccaactaaat gggcactcga gttagccctc ccacacataa ccagagggca
     3181 gcaattcacg aatcccaact gccgtcggct gtccatcact gtccttcact atggctttga
     3241 tcccaggatg cagatcgaga agcacctgtc ggcaccgtcc gcaggggctc aagatgcccc
     3301 tgttctcatt tccgatcgcg acgatacaag tcaggttgcc agctgccgca gcagcagcag
     3361 tgcccagcac cacgagttct gcacaaggtc ccccagtaaa atgatataca ttgacaccag
     3421 tgaagatgcg gccgtcgcta gagagagctg cgctggcgac gctgtagtct tcagagatgg
     3481 ggatgctgtt gattgtagcc gttgctcttt caatgagggt ggattcttct tgagacaaag
     3541 gcatgctagc tgatgcttta ttgctttctc ttaaatttgg gataaccgta gtttatgcgg
     3601 cagtagtttg acaccttttc gagggggaaa gagacaaaaa aggagaagac aaggattaaa
     3661 gggtgcaccc aaaggaggaa acacacggtt ggtgtgcagt gatgaatatc aagttgcaca
     3721 acagtgcaga ctcaaaacgg cactaatgaa ccagatgtca gacatgcagc tacgatctac
     3781 acaaacagtg actctctaat gccgaaggat aggcaccgca gtgcacgtac acagcggcat
     3841 agattgaaat actacataaa ggttatacat tgattataat gaaagacatt taagcgcagc
     3901 ggagctctta actacccgat cctgatccag atcctgatcc ggatccgagg tgagcctcca
     3961 atgtctcgga ctggcaaatg tgatcccttt cttcagtatc gtctttcgac tgcgtatact
     4021 gtttgcgaat ccaatgatag ggcacatcgg gagccggctg attgtgtaac ggcttacaga
     4081 ttgtattttg atgagcctcg acgcacttag acttatcact aagaagcggg tatagaaggg
     4141 tgacggggca ttccacccca tcgtcgcgtg ggatatgctg cacatcattt ggtaagctca
     4201 cattcatatc gcccgtaagt accggactgt tttctttaaa gtcttgccct gtgaattcaa
     4261 ctatgttatg atatgtgccg ttcttcatga agtgctgatg tttcgcatgt atggaaccat
     4321 cgcccttata ttgaatgtgg cgatcgtatg taaaaccttc tggcatgact tcgtgaaacc
     4381 agtttttcag acccgaagga tacttcgtgt agtatttcat tccgtatccg aaagacgtgc
     4441 ccaatgccgc ccatgacata ggtaatttac cagaggtgca gacgtattta cccttgtgcg
     4501 atccctcgtg cgaatttcct tcaccttcac cctccacggt aaaagacttg ccgttgatag
     4561 ttcccttcaa ctgaaacttg aagggtgtgg atgcaagctt agaaccggaa ccggaaccac
     4621 taccagaacc cataggtacc cgttgcagtt gagtttctgt tttcgggctt gttggtatac
     4681 ttgtttgatt aatgtggctg atagctaagc tatcgcaaat atttgtacaa gacatgaggg
     4741 ggcaaaaaaa taagccaagt gtatttgtta ggcttccgtg cctgttagag aaatatgtaa
     4801 ggaaacgcct gcgctacaca gcttgagcaa cgcgtggggc tcgaatcccc ccattttctt
     4861 cttttacatc atcccccctt cctaaccaac ctgcaggcgc acctccctgc tgtgccatca
     4921 gattactccg ggaacctttc cacggaaaag acacgagaca agggaaaggc caactaaatg
     4981 ggcaccatgg ttagccctcc cacacataac cagagggcag caattcacga atcccaactg
     5041 ccgtcggctg tccatcactg tccttcacta tggctttgat cccaggatgc aggtcgagaa
     5101 gcacctgtcg gcaccgtccg caggggctca agatgcccct gttctcattt ccgatcgcga
     5161 cgatacaagt caggttgcca gctgccgcag cagcagcagt gcccagcacc acgagttctg
     5221 cacaaggtcc cccagtaaaa tgatatacat tgacaccagt gaagatgcgg ccgtcgctag
     5281 agagagctgc gctggcgacg ctgtagtctt cagagatggg gatgctgttg attgtagccg
     5341 ttgctctttc aatgagggtg gattcttctt gagacaaagg catgaattcc actagagctt
     5401 attttatggc agcaacgaga ccttacgtaa agcaaaacta aataagagcg gagactgcaa
     5461 tgcagagtaa
//
