LOCUS       sequence-355744-        5542 bp    DNA     circular SYN 14-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 5542)
  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 5542)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-eGFP
FEATURES             Location/Qualifiers
     source          1..5542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(960..962,963..1673)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGEVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1263..1282)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1610..1631
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2667..2685)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2668..2685)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2703..2719)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2703..2719)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2716..2738)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2727..2743
                     /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(2751..2757,2758..2775,2776..2781))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2796..2817
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2934..2951)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3105..3693)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3185..3204)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3864..4655,4656..4724))
                     /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     4487..4506
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4725..4829)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4907..5362)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5044..5065)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5256..5275
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5488..5510
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5502..5519
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5503..5519
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5526..5542,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5526..5542,1..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 cgaggagctg ttcaccgggg tggtgcccat actggtcgag ctggacggcg
     1021 aagtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1081 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1141 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     1201 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1261 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1321 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1381 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     1441 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1501 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1561 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1621 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagcggtacg
     1681 gatccggatc aggatctgga tcaggatcgg gtagttaaga gctccgctgc gcttaaatgt
     1741 ctttcattat aatcaatgta taacctttat gtagtatttc aatctatgcc gctgtgtacg
     1801 tgcactgcgg tgcctatcct tcggcattag agagtcactg tttgtgtaga tcgtagctgc
     1861 atgtctgaca tctggttcat tagtgccgtt ttgagtctgc actgttgtgc aacttgatat
     1921 tcatcactgc acaccaaccg tgtgtttcct cctttgggtg caccctttaa tccttgtctt
     1981 ctcctttttt gtctctttcc ccctcgaaaa ggtgtcaaac tactgccgca taaactacgg
     2041 ttatcccaaa tttaagagaa agcaataaag catcagctag catgcctttg tctcaagaag
     2101 aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact
     2161 acagcgtcgc cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat
     2221 atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg
     2281 cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc
     2341 cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga
     2401 aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg
     2461 tgtgggaggg ctaactcgag tgcccattta gttggctttt cccttgtctc gtgtcttttc
     2521 cgtggaaagg ttcccggagt aatctgatgg cacagcaggg aggtgcgcct gcaggttggt
     2581 taggaagggg ggatgatgta aaagaagaaa atggggggat tcgagccccg cacaggtctc
     2641 tcaaattgga tgcatagctt gagtattcta tagtgtcacc taaatagctt ggcgtaatca
     2701 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     2761 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     2821 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2881 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2941 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3001 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3061 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3121 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3181 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3241 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3301 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3361 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3421 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3481 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3541 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3601 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3661 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3721 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3781 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3841 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3901 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aattacgata
     3961 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4021 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4081 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4141 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4201 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4261 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4321 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4381 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4441 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4501 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4561 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4621 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4681 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4741 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4801 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg
     4861 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt
     4921 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     4981 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     5041 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     5101 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     5161 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     5221 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     5281 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcactt
     5341 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc
     5401 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     5461 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     5521 aattgtaata cgactcacta ta
//
