LOCUS       sequence-374796-        6802 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 6802)
  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 6802)
  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-hygro-eYFP
FEATURES             Location/Qualifiers
     source          1..6802
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             171..1196
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             171..1196
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             join(1593..1595,1596..2306)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1635..1656)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1896..1915)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2243..2264
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2709..3734
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2709..3734
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(3927..3945)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3928..3945)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3963..3979)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3963..3979)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3976..3998)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3987..4003
                     /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(4011..4017,4018..4035,4036..4041))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4056..4077
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4194..4211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4365..4953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4445..4464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5124..5915,5916..5984))
                     /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     5747..5766
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5985..6089)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6167..6622)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6304..6325)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6516..6535
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6748..6770
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6762..6779
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6763..6779
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6786..6802,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6786..6802,1..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 atgaaaaagc
      181 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
      241 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
      301 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
      361 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattta
      421 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
      481 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggcaatggat gcgatcgctg
      541 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
      601 acactacatg gcgtgatttt atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
      661 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
      721 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
      781 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
      841 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
      901 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgcct cggctccggg
      961 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     1021 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     1081 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     1141 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagccat
     1201 ggtgcccatt tagttggcct ttcccttgtc tcgtgtcttt tccgtggaaa ggttcccgga
     1261 gtaatctgat ggcacagcag ggaggtgcgc ctgcaggttg gttaggaagg ggggatgatg
     1321 taaaagaaga aaatgggggg attcgagccc cacgcgttgc tcaagctgtg tagcgcaggc
     1381 gtttccttac atatttctct aacaggcacg gaagcctaac aaatacactt ggcttatttt
     1441 tttgccccct catgtcttgt acaaatattt gcgatagctt agctatcagc cacattaatc
     1501 aaacaagtat accaacaagc ccgaaaacag aaactcaact gcaacgggta cctatgggtt
     1561 ctggtagtgg ttccggttcc ggttctaagc ttgtgagcaa gggcgaggag ctgttcaccg
     1621 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1681 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1741 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgcagt
     1801 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1861 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1921 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1981 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2041 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2101 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2161 acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc ctgagcaaag
     2221 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2281 ctctcggcat ggacgagctg tacaagggat ccggatcagg atctggatca ggatcgggta
     2341 gttaagagct ccgctgcgct taaatgtctt tcattataat caatgtataa cctttatgta
     2401 gtatttcaat ctatgccgct gtgtacgtgc actgcggtgc ctatccttcg gcattagaga
     2461 gtcactgttt gtgtagatcg tagctgcatg tctgacatct ggttcattag tgccgttttg
     2521 agtctgcact gttgtgcaac ttgatattca tcactgcaca ccaaccgtgt gtttcctcct
     2581 ttgggtgcac cctttaatcc ttgtcttctc cttttttgtc tctttccccc tcgaaaaggt
     2641 gtcaaactac tgccgcataa actacggtta tcccaaattt aagagaaagc aataaagcat
     2701 cagctagcat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa
     2761 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca
     2821 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
     2881 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
     2941 ttgacattgg ggaatttagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
     3001 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
     3061 caatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
     3121 cgcaaggaat cggtcaatac actacatggc gtgattttat atgcgcgatt gctgatcccc
     3181 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
     3241 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
     3301 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
     3361 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
     3421 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
     3481 gatcgcctcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
     3541 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
     3601 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga
     3661 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
     3721 gggcaaagga atagctcgag tgcccattta gttggctttt cccttgtctc gtgtcttttc
     3781 cgtggaaagg ttcccggagt aatctgatgg cacagcaggg aggtgcgcct gcaggttggt
     3841 taggaagggg ggatgatgta aaagaagaaa atggggggat tcgagccccg cacaggtctc
     3901 tcaaattgga tgcatagctt gagtattcta tagtgtcacc taaatagctt ggcgtaatca
     3961 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4021 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4081 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4141 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4201 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4261 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4321 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4381 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4441 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4501 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4561 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4621 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4681 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4741 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4801 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4861 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4921 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4981 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5041 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5101 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5161 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aattacgata
     5221 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5281 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5341 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5401 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5461 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5521 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5581 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5641 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5701 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5761 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5821 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5881 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5941 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6001 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6061 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg
     6121 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt
     6181 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     6241 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     6301 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     6361 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     6421 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     6481 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     6541 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcactt
     6601 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc
     6661 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     6721 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     6781 aattgtaata cgactcacta ta
//
