LOCUS       sequence-374799-        6727 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 6727)
  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 6727)
  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-mNG2(1-10)
FEATURES             Location/Qualifiers
     source          1..6727
                     /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             2634..3659
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2634..3659
                     /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(3852..3870)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3853..3870)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3888..3904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3888..3904)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3901..3923)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3912..3928
                     /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(3936..3942,3943..3960,3961..3966))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3981..4002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4119..4136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4290..4878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4370..4389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5049..5840,5841..5909))
                     /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     5672..5691
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5910..6014)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6092..6547)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6229..6250)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6441..6460
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6673..6695
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6687..6704
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6688..6704
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6711..6727,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6711..6727,1..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 gaagggtgaa gaagataata
     1621 tggcttccct tcccgcgacg cacgagctac atatcttcgg cagtataaat ggggtcgact
     1681 tcgacatggt tggtcaaggg acgggtaacc ctaacgacgg atatgaagaa ctcaatctca
     1741 agagcacaaa gggcgacctg cagtttagtc cctggattct cgtgcctcat attggttatg
     1801 gctttcatca atacctaccg tatcccgatg gtatgagccc gttccaagca gcgatggtag
     1861 acggatcagg atatcaggtc catcgtacta tgcaatttga agacggcgca tccctgacgg
     1921 ttaactaccg gtatacttac gaaggaagcc acattaaggg ggaagcccag gttatgggca
     1981 ctggtttccc agcggatgga ccagttatga caaacacact taccgccgcg gattggtgca
     2041 tgtcaaagaa gacgtatcct aatgacaaaa cgatcatatc gaccttcaag tggtcctaca
     2101 caactgtgaa cggaaagcgg tatcggtcaa cagcgcggac cacctacact ttcgctaagc
     2161 caatggctgc gaactacctc aaaaatcagc ccatgtatgt ttttcggaag acagagttga
     2221 agcacagtat gggatccgga tcaggatctg gatcaggatc gggtagttaa gagctccgct
     2281 gcgcttaaat gtctttcatt ataatcaatg tataaccttt atgtagtatt tcaatctatg
     2341 ccgctgtgta cgtgcactgc ggtgcctatc cttcggcatt agagagtcac tgtttgtgta
     2401 gatcgtagct gcatgtctga catctggttc attagtgccg ttttgagtct gcactgttgt
     2461 gcaacttgat attcatcact gcacaccaac cgtgtgtttc ctcctttggg tgcacccttt
     2521 aatccttgtc ttctcctttt ttgtctcttt ccccctcgaa aaggtgtcaa actactgccg
     2581 cataaactac ggttatccca aatttaagag aaagcaataa agcatcagct agcatgaaaa
     2641 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     2701 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     2761 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     2821 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     2881 ttagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     2941 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggcaatg gatgcgatcg
     3001 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     3061 aatacactac atggcgtgat tttatatgcg cgattgctga tccccatgtg tatcactggc
     3121 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     3181 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     3241 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     3301 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     3361 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg cctcggctcc
     3421 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     3481 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     3541 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     3601 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     3661 tcgagtgccc atttagttgg cttttccctt gtctcgtgtc ttttccgtgg aaaggttccc
     3721 ggagtaatct gatggcacag cagggaggtg cgcctgcagg ttggttagga aggggggatg
     3781 atgtaaaaga agaaaatggg gggattcgag ccccgcacag gtctctcaaa ttggatgcat
     3841 agcttgagta ttctatagtg tcacctaaat agcttggcgt aatcatggtc atagctgttt
     3901 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3961 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4021 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4081 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4141 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4201 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4261 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4321 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4381 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4441 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4501 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4561 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4621 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4681 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4741 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4801 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4861 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4921 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4981 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5041 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5101 tcatccatag ttgcctgact ccccgtcgtg tagataatta cgatacggga gggcttacca
     5161 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5221 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5281 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5341 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5401 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5461 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5521 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5581 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5641 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5701 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5761 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5821 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5881 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5941 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6001 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg atgcggtgtg aaataccgca
     6061 cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa
     6121 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     6181 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     6241 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     6301 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     6361 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     6421 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     6481 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cacttaatgc gccgctacag
     6541 ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     6601 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     6661 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact
     6721 cactata
//
