LOCUS       sequence-400582-        6988 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 6988)
  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 6988)
  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: pPOTv6-hygro-3Ty:mScarletI:3Ty
FEATURES             Location/Qualifiers
     source          1..6988
                     /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             2895..3920
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2895..3920
                     /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(4113..4131)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4114..4131)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4149..4165)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4149..4165)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4162..4184)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4173..4189
                     /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(4197..4203,4204..4221,4222..4227))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4242..4263
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4380..4397)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4551..5139)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4631..4650)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5310..6101,6102..6170))
                     /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     5933..5952
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6171..6275)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6353..6808)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6490..6511)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6702..6721
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6934..6956
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6948..6965
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6949..6965
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6972..6988,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6972..6988,1..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 ggttctgagg tacacacaaa ccaggaccca ctggacggct
     1621 ccgaggtgca taccaatcaa gatccgctgg acggctctga agttcacacc aatcaagatc
     1681 ccttggataa gcttatggta tcaaagggcg aagcagtaat caaggagttc atgcggttta
     1741 aggtacacat ggagggttcc atgaatggcc atgaatttga gatcgaagga gagggggaag
     1801 ggcgtcctta cgaaggaaca caaacagcta aacttaaggt aactaagggg ggacccctcc
     1861 ccttttcctg ggacatactg tcaccacaat tcatgtacgg cagccgagcc tttataaaac
     1921 atccagctga tatacccgat tattataagc aaagttttcc cgagggcttt aagtgggaaa
     1981 gggtcatgaa cttcgaggac gggggtgcgg taactgtaac ccaagacacc tctctcgaag
     2041 acgggacgct tatttataag gtgaaactcc gggggaccaa ttttccccct gacggacctg
     2101 tcatgcagaa gaagacaatg ggctgggagg cgtccacaga gcgcctgtat ccggaggatg
     2161 gcgtacttaa gggtgacata aaaatggcac ttcgtttgaa ggacggtggc cgttatctcg
     2221 ctgactttaa gacaacttat aaggcgaaaa agccggtaca gatgccagga gcatacaatg
     2281 ttgaccgcaa attggacatc accagccaca acgaagacta taccgtcgtt gaacagtacg
     2341 aacggagtga agggaggcac agtacaggcg ggatggacga attgtataaa ggatccgagg
     2401 tacatacaaa tcaggacccc ctcgatggta gtgaagtcca tacaaatcag gacccgctgg
     2461 atggctcaga ggttcatacg aaccaagatc ctcttgatgg atcaggatct ggatcaggat
     2521 cgggtagtta agagctccgc tgcgcttaaa tgtctttcat tataatcaat gtataacctt
     2581 tatgtagtat ttcaatctat gccgctgtgt acgtgcactg cggtgcctat ccttcggcat
     2641 tagagagtca ctgtttgtgt agatcgtagc tgcatgtctg acatctggtt cattagtgcc
     2701 gttttgagtc tgcactgttg tgcaacttga tattcatcac tgcacaccaa ccgtgtgttt
     2761 cctcctttgg gtgcaccctt taatccttgt cttctccttt tttgtctctt tccccctcga
     2821 aaaggtgtca aactactgcc gcataaacta cggttatccc aaatttaaga gaaagcaata
     2881 aagcatcagc tagcatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     2941 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     3001 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     3061 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     3121 aagtgcttga cattggggaa tttagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     3181 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     3241 cggaggcaat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     3301 tcggaccgca aggaatcggt caatacacta catggcgtga ttttatatgc gcgattgctg
     3361 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     3421 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     3481 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     3541 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     3601 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     3661 ttgcaggatc gcctcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     3721 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     3781 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     3841 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     3901 gtccgagggc aaaggaatag ctcgagtgcc catttagttg gcttttccct tgtctcgtgt
     3961 cttttccgtg gaaaggttcc cggagtaatc tgatggcaca gcagggaggt gcgcctgcag
     4021 gttggttagg aaggggggat gatgtaaaag aagaaaatgg ggggattcga gccccgcaca
     4081 ggtctctcaa attggatgca tagcttgagt attctatagt gtcacctaaa tagcttggcg
     4141 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4201 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4261 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4321 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4381 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4441 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4501 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4561 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4621 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4681 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4741 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4801 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4861 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4921 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4981 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5041 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5101 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5161 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5221 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5281 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5341 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataatt
     5401 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5461 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5521 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5581 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5641 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5701 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5761 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5821 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5881 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5941 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6001 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6061 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6121 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6181 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6241 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6301 gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta
     6361 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     6421 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     6481 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     6541 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     6601 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     6661 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     6721 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     6781 gcacttaatg cgccgctaca gggcgcgtcc attcgccatt caggctgcgc aactgttggg
     6841 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     6901 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     6961 ccagtgaatt gtaatacgac tcactata
//
