LOCUS       sequence-355698-        6784 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 6784)
  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 6784)
  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-mScarletI
FEATURES             Location/Qualifiers
     source          1..6784
                     /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             2691..3716
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2691..3716
                     /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(3909..3927)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3910..3927)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3945..3961)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3945..3961)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3958..3980)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3969..3985
                     /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(3993..3999,4000..4017,4018..4023))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4038..4059
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4176..4193)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4347..4935)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4427..4446)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5106..5897,5898..5966))
                     /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     5729..5748
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5967..6071)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6149..6604)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6286..6307)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6498..6517
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6730..6752
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6744..6761
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6745..6761
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6768..6784,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6768..6784,1..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 ttatggtatc aaagggcgaa gcagtaatca
     1621 aggagttcat gcggtttaag gtacacatgg agggttccat gaatggccat gaatttgaga
     1681 tcgaaggaga gggggaaggg cgtccttacg aaggaacaca aacagctaaa cttaaggtaa
     1741 ctaagggggg acccctcccc ttttcctggg acatactgtc accacaattc atgtacggca
     1801 gccgagcctt tataaaacat ccagctgata tacccgatta ttataagcaa agttttcccg
     1861 agggctttaa gtgggaaagg gtcatgaact tcgaggacgg gggtgcggta actgtaaccc
     1921 aagacacctc tctcgaagac gggacgctta tttataaggt gaaactccgg gggaccaatt
     1981 ttccccctga cggacctgtc atgcagaaga agacaatggg ctgggaggcg tccacagagc
     2041 gcctgtatcc ggaggatggc gtacttaagg gtgacataaa aatggcactt cgtttgaagg
     2101 acggtggccg ttatctcgct gactttaaga caacttataa ggcgaaaaag ccggtacaga
     2161 tgccaggagc atacaatgtt gaccgcaaat tggacatcac cagccacaac gaagactata
     2221 ccgtcgttga acagtacgaa cggagtgaag ggaggcacag tacaggcggg atggacgaat
     2281 tgtataaagg atccggatca ggatctggat caggatcggg tagttaagag ctccgctgcg
     2341 cttaaatgtc tttcattata atcaatgtat aacctttatg tagtatttca atctatgccg
     2401 ctgtgtacgt gcactgcggt gcctatcctt cggcattaga gagtcactgt ttgtgtagat
     2461 cgtagctgca tgtctgacat ctggttcatt agtgccgttt tgagtctgca ctgttgtgca
     2521 acttgatatt catcactgca caccaaccgt gtgtttcctc ctttgggtgc accctttaat
     2581 ccttgtcttc tccttttttg tctctttccc cctcgaaaag gtgtcaaact actgccgcat
     2641 aaactacggt tatcccaaat ttaagagaaa gcaataaagc atcagctagc atgaaaaagc
     2701 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
     2761 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
     2821 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
     2881 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattta
     2941 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
     3001 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggcaatggat gcgatcgctg
     3061 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
     3121 acactacatg gcgtgatttt atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     3181 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     3241 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     3301 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     3361 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     3421 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgcct cggctccggg
     3481 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     3541 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     3601 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     3661 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagctcg
     3721 agtgcccatt tagttggctt ttcccttgtc tcgtgtcttt tccgtggaaa ggttcccgga
     3781 gtaatctgat ggcacagcag ggaggtgcgc ctgcaggttg gttaggaagg ggggatgatg
     3841 taaaagaaga aaatgggggg attcgagccc cgcacaggtc tctcaaattg gatgcatagc
     3901 ttgagtattc tatagtgtca cctaaatagc ttggcgtaat catggtcata gctgtttcct
     3961 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4021 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4081 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4141 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4201 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4261 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4321 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4381 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4441 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4501 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4561 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4621 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4681 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4741 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4801 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4861 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4921 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4981 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5041 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5101 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5161 tccatagttg cctgactccc cgtcgtgtag ataattacga tacgggaggg cttaccatct
     5221 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5281 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5341 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5401 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5461 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5521 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5581 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5641 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5701 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5761 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5821 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5881 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5941 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6001 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6061 ggggttccgc gcacatttcc ccgaaaagtg ccacctgatg cggtgtgaaa taccgcacag
     6121 atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg ttaatatttt gttaaaattc
     6181 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     6241 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     6301 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     6361 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     6421 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     6481 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     6541 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcac ttaatgcgcc gctacagggc
     6601 gcgtccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     6661 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     6721 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac
     6781 tata
//
