LOCUS       sequence-374355-        5710 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 5710)
  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 5710)
  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-HaloTag
FEATURES             Location/Qualifiers
     source          1..5710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             960..1847
                     /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(2835..2853)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2836..2853)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2871..2887)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2871..2887)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2884..2906)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2895..2911
                     /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(2919..2925,2926..2943,2944..2949))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2964..2985
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3102..3119)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3273..3861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3353..3372)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4032..4823,4824..4892))
                     /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     4655..4674
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4893..4997)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5075..5530)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5212..5233)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5424..5443
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5656..5678
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5670..5687
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5671..5687
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5694..5710,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5694..5710,1..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 ctgagattgg tacggggttt ccttttgacc cgcactacgt tgaggtgttg ggtgaacgta
     1021 tgcattatgt tgatgttggt ccccgtgatg gtacgcccgt gctgtttttg catggtaacc
     1081 ccacgtcaag ctatgtgtgg aggaacatta ttccccacgt ggcacctact catcgctgta
     1141 ttgctccaga ccttattggt atgggtaagt cagataaacc ggatcttggt tacttctttg
     1201 acgatcacgt gcgttttatg gatgcattta ttgaagcgct tggccttgaa gaagttgtgt
     1261 tagtgattca cgattggggt tctgccttgg gatttcattg ggctaaacgt aatcccgaaa
     1321 gagttaaagg tattgcattc atggagttca ttcgtcctat tcctacgtgg gatgagtggc
     1381 cagagtttgc gcgcgagacg tttcaagcgt ttcgtacaac ggacgtgggt cgtaagctga
     1441 ttattgatca aaatgttttt attgagggta cgctgccgat gggtgttgtg cgtcccctga
     1501 ctgaagtgga gatggatcac taccgtgagc catttttaaa tccagtggac cgtgagccat
     1561 tgtggcgctt ccctaacgag ctgcccattg ctggtgaacc ggctaacatt gtggcgcttg
     1621 tggaggagta tatggattgg cttcatcaat cacccgtgcc taagttgctt ttttggggta
     1681 cgccaggtgt gcttattcca cctgctgaag cagcacgctt ggctaaatca cttccaaact
     1741 gcaaagcagt tgatattggt ccgggtctga acctgttgca agaagataat cccgatctta
     1801 ttggttccga gattgcccgt tggctttcga cacttgaaat ttctggtgga tccggatcag
     1861 gatctggatc aggatcgggt agttaagagc tccgctgcgc ttaaatgtct ttcattataa
     1921 tcaatgtata acctttatgt agtatttcaa tctatgccgc tgtgtacgtg cactgcggtg
     1981 cctatccttc ggcattagag agtcactgtt tgtgtagatc gtagctgcat gtctgacatc
     2041 tggttcatta gtgccgtttt gagtctgcac tgttgtgcaa cttgatattc atcactgcac
     2101 accaaccgtg tgtttcctcc tttgggtgca ccctttaatc cttgtcttct ccttttttgt
     2161 ctctttcccc ctcgaaaagg tgtcaaacta ctgccgcata aactacggtt atcccaaatt
     2221 taagagaaag caataaagca tcagctagca tgcctttgtc tcaagaagaa tccaccctca
     2281 ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca
     2341 gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg
     2401 ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc
     2461 tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt
     2521 gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg
     2581 gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct
     2641 aactcgagtg cccatttagt tggcttttcc cttgtctcgt gtcttttccg tggaaaggtt
     2701 cccggagtaa tctgatggca cagcagggag gtgcgcctgc aggttggtta ggaagggggg
     2761 atgatgtaaa agaagaaaat ggggggattc gagccccgca caggtctctc aaattggatg
     2821 catagcttga gtattctata gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg
     2881 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     2941 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     3001 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3061 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3121 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3181 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3241 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3301 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3361 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3421 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3481 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3541 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3601 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3661 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3721 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3781 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3841 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3901 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3961 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4021 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4081 cgttcatcca tagttgcctg actccccgtc gtgtagataa ttacgatacg ggagggctta
     4141 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4201 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4261 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4321 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4381 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4441 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4501 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4561 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4621 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4681 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4741 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4801 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4861 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4921 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4981 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgatgcggt gtgaaatacc
     5041 gcacagatgc gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta
     5101 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     5161 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     5221 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     5281 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     5341 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     5401 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     5461 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcacttaa tgcgccgcta
     5521 cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     5581 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     5641 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg
     5701 actcactata
//
