LOCUS       sequence-355742-        6424 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 6424)
  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 6424)
  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-blast-mNG:HaloTag
FEATURES             Location/Qualifiers
     source          1..6424
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1674..2561
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     promoter        complement(3549..3567)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3550..3567)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3585..3601)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3585..3601)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3598..3620)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3609..3625
                     /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(3633..3639,3640..3657,3658..3663))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3678..3699
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3816..3833)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3987..4575)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4067..4086)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4746..5537,5538..5606))
                     /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     5369..5388
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5607..5711)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5789..6244)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5926..5947)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6138..6157
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6370..6392
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6384..6401
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6385..6401
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6408..6424,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6408..6424,1..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 tctcgaaagg tgaggaagac aatatggcca gcttgccagc aacgcacgag cttcatatat
     1021 ttgggtcgat caacggtgtg gatttcgaca tggtgggtca aggcacaggt aatcctaatg
     1081 acgggtatga agaactcaat ttgaagagca ctaagggaga tctccagttt tccccgtgga
     1141 ttcttgtgcc gcatattggt tatggttttc atcagtactt accatacccc gatggaatgt
     1201 cgcccttcca agcagctatg gtggacggct cggggtatca agtacatcgc accatgcaat
     1261 ttgaggacgg agcatccctc accgtaaact accgctatac gtatgagggg agccacataa
     1321 aaggtgaggc tcaagttaaa ggcacaggct tcccagctga cggcccggtc atgaccaatt
     1381 cattaaccgc tgctgattgg tgccgttcta agaagaccta ccctaacgac aagacgatca
     1441 ttagtacttt taagtggagt tataccacag gaaacggaaa gcggtatcga agcactgctc
     1501 gaacaaccta cacatttgcg aagccaatgg ccgcgaacta cttgaagaac cagccaatgt
     1561 atgtgtttcg aaaaacggaa ctgaagcatt cgaaaaccga gcttaatttt aaagagtggc
     1621 agaaggcgtt tacagacgtg atggggatgg acgaattgta taagcccggg tcggctgaga
     1681 ttggtacggg gtttcctttt gacccgcact acgttgaggt gttgggtgaa cgtatgcatt
     1741 atgttgatgt tggtccccgt gatggtacgc ccgtgctgtt tttgcatggt aaccccacgt
     1801 caagctatgt gtggaggaac attattcccc acgtggcacc tactcatcgc tgtattgctc
     1861 cagaccttat tggtatgggt aagtcagata aaccggatct tggttacttc tttgacgatc
     1921 acgtgcgttt tatggatgca tttattgaag cgcttggcct tgaagaagtt gtgttagtga
     1981 ttcacgattg gggttctgcc ttgggatttc attgggctaa acgtaatccc gaaagagtta
     2041 aaggtattgc attcatggag ttcattcgtc ctattcctac gtgggatgag tggccagagt
     2101 ttgcgcgcga gacgtttcaa gcgtttcgta caacggacgt gggtcgtaag ctgattattg
     2161 atcaaaatgt ttttattgag ggtacgctgc cgatgggtgt tgtgcgtccc ctgactgaag
     2221 tggagatgga tcactaccgt gagccatttt taaatccagt ggaccgtgag ccattgtggc
     2281 gcttccctaa cgagctgccc attgctggtg aaccggctaa cattgtggcg cttgtggagg
     2341 agtatatgga ttggcttcat caatcacccg tgcctaagtt gcttttttgg ggtacgccag
     2401 gtgtgcttat tccacctgct gaagcagcac gcttggctaa atcacttcca aactgcaaag
     2461 cagttgatat tggtccgggt ctgaacctgt tgcaagaaga taatcccgat cttattggtt
     2521 ccgagattgc ccgttggctt tcgacacttg aaatttctgg tggatccgga tcaggatctg
     2581 gatcaggatc gggtagttaa gagctccgct gcgcttaaat gtctttcatt ataatcaatg
     2641 tataaccttt atgtagtatt tcaatctatg ccgctgtgta cgtgcactgc ggtgcctatc
     2701 cttcggcatt agagagtcac tgtttgtgta gatcgtagct gcatgtctga catctggttc
     2761 attagtgccg ttttgagtct gcactgttgt gcaacttgat attcatcact gcacaccaac
     2821 cgtgtgtttc ctcctttggg tgcacccttt aatccttgtc ttctcctttt ttgtctcttt
     2881 ccccctcgaa aaggtgtcaa actactgccg cataaactac ggttatccca aatttaagag
     2941 aaagcaataa agcatcagct agcatgcctt tgtctcaaga agaatccacc ctcattgaaa
     3001 gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag
     3061 ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac
     3121 cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt
     3181 gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac
     3241 aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc
     3301 cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaactcg
     3361 agtgcccatt tagttggctt ttcccttgtc tcgtgtcttt tccgtggaaa ggttcccgga
     3421 gtaatctgat ggcacagcag ggaggtgcgc ctgcaggttg gttaggaagg ggggatgatg
     3481 taaaagaaga aaatgggggg attcgagccc cgcacaggtc tctcaaattg gatgcatagc
     3541 ttgagtattc tatagtgtca cctaaatagc ttggcgtaat catggtcata gctgtttcct
     3601 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     3661 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3721 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3781 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3841 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3901 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3961 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4021 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4081 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4141 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4201 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4261 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4321 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4381 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4441 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4501 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4561 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4621 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4681 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4741 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4801 tccatagttg cctgactccc cgtcgtgtag ataattacga tacgggaggg cttaccatct
     4861 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4921 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4981 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5041 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5101 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5161 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5221 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5281 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5341 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5401 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5461 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5521 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5581 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5641 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5701 ggggttccgc gcacatttcc ccgaaaagtg ccacctgatg cggtgtgaaa taccgcacag
     5761 atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg ttaatatttt gttaaaattc
     5821 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     5881 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     5941 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     6001 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     6061 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     6121 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     6181 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcac ttaatgcgcc gctacagggc
     6241 gcgtccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     6301 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     6361 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac
     6421 tata
//
