LOCUS       sequence-400048-        6136 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 6136)
  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 6136)
  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: pPOTv6-puro-3Ty:mScarletI:3Ty
FEATURES             Location/Qualifiers
     source          1..6136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(3261..3279)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3262..3279)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3297..3313)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3297..3313)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3310..3332)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3321..3337
                     /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(3345..3351,3352..3369,3370..3375))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3390..3411
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3528..3545)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3699..4287)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3779..3798)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4458..5249,5250..5318))
                     /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     5081..5100
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5319..5423)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5501..5956)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5638..5659)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5850..5869
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6082..6104
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6096..6113
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6097..6113
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6120..6136,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6120..6136,1..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 atgactgaat
      181 acaagccaac ggttcgtctt gctacgcgag acgatgtgcc gcgggccgta cggacgctgg
      241 ctgctgcgtt cgctgattat cccgccaccc gacatacggt tgacccagat cgacacattg
      301 agcgagtcac agaacttcaa gagttattct taacacgggt gggcttggac attggtaagg
      361 tttgggttgc ggacgatggc gcggctgttg cggtgtggac gacgccagag tcagtcgaag
      421 cgggcgcagt gtttgctgag atcggtcccc gaatggcgga gcttagcggg tcatgcctcg
      481 ctgcacaaca gcagatggaa ggtctgttgg caccacatag acctaaggag ccggcttggt
      541 tcttagcgac agttggcgtc agtccggacc accaaggtaa ggggctgggt tcagcggttg
      601 ttcttccagg agtagaggct gcggaaaggg caggtgtgcc ggcgttcctt gaaacgtctg
      661 caccacgtaa ccttcctttt tacgagagat tggggtttac agtaacagca gatgttgaag
      721 tgcctgaagg gccgaggaca tggtgcatga cacgtaagcc gggagcctaa ccatggtgcc
      781 catttagttg gcctttccct tgtctcgtgt cttttccgtg gaaaggttcc cggagtaatc
      841 tgatggcaca gcagggaggt gcgcctgcag gttggttagg aaggggggat gatgtaaaag
      901 aagaaaatgg ggggattcga gccccacgcg ttgctcaagc tgtgtagcgc aggcgtttcc
      961 ttacatattt ctctaacagg cacggaagcc taacaaatac acttggctta tttttttgcc
     1021 ccctcatgtc ttgtacaaat atttgcgata gcttagctat cagccacatt aatcaaacaa
     1081 gtataccaac aagcccgaaa acagaaactc aactgcaacg ggtacctatg ggttctggta
     1141 gtggttccgg ttccggttct gaggtacaca caaaccagga cccactggac ggctccgagg
     1201 tgcataccaa tcaagatccg ctggacggct ctgaagttca caccaatcaa gatcccttgg
     1261 ataagcttat ggtatcaaag ggcgaagcag taatcaagga gttcatgcgg tttaaggtac
     1321 acatggaggg ttccatgaat ggccatgaat ttgagatcga aggagagggg gaagggcgtc
     1381 cttacgaagg aacacaaaca gctaaactta aggtaactaa ggggggaccc ctcccctttt
     1441 cctgggacat actgtcacca caattcatgt acggcagccg agcctttata aaacatccag
     1501 ctgatatacc cgattattat aagcaaagtt ttcccgaggg ctttaagtgg gaaagggtca
     1561 tgaacttcga ggacgggggt gcggtaactg taacccaaga cacctctctc gaagacggga
     1621 cgcttattta taaggtgaaa ctccggggga ccaattttcc ccctgacgga cctgtcatgc
     1681 agaagaagac aatgggctgg gaggcgtcca cagagcgcct gtatccggag gatggcgtac
     1741 ttaagggtga cataaaaatg gcacttcgtt tgaaggacgg tggccgttat ctcgctgact
     1801 ttaagacaac ttataaggcg aaaaagccgg tacagatgcc aggagcatac aatgttgacc
     1861 gcaaattgga catcaccagc cacaacgaag actataccgt cgttgaacag tacgaacgga
     1921 gtgaagggag gcacagtaca ggcgggatgg acgaattgta taaaggatcc gaggtacata
     1981 caaatcagga ccccctcgat ggtagtgaag tccatacaaa tcaggacccg ctggatggct
     2041 cagaggttca tacgaaccaa gatcctcttg atggatcagg atctggatca ggatcgggta
     2101 gttaagagct ccgctgcgct taaatgtctt tcattataat caatgtataa cctttatgta
     2161 gtatttcaat ctatgccgct gtgtacgtgc actgcggtgc ctatccttcg gcattagaga
     2221 gtcactgttt gtgtagatcg tagctgcatg tctgacatct ggttcattag tgccgttttg
     2281 agtctgcact gttgtgcaac ttgatattca tcactgcaca ccaaccgtgt gtttcctcct
     2341 ttgggtgcac cctttaatcc ttgtcttctc cttttttgtc tctttccccc tcgaaaaggt
     2401 gtcaaactac tgccgcataa actacggtta tcccaaattt aagagaaagc aataaagcat
     2461 cagctagcat gactgaatac aagccaacgg ttcgtcttgc tacgcgagac gatgtgccgc
     2521 gggccgtacg gacgctggct gctgcgttcg ctgattatcc cgccacccga catacggttg
     2581 acccagatcg acacattgag cgagtcacag aacttcaaga gttattctta acacgggtgg
     2641 gcttggacat tggtaaggtt tgggttgcgg acgatggcgc ggctgttgcg gtgtggacga
     2701 cgccagagtc agtcgaagcg ggcgcagtgt ttgctgagat cggtccccga atggcggagc
     2761 ttagcgggtc acgcctcgct gcacaacagc agatggaagg tctgttggca ccacatagac
     2821 ctaaggagcc ggcttggttc ttagcgacag ttggcgtcag tccggaccac caaggtaagg
     2881 ggctgggttc agcggttgtt cttccaggag tagaggctgc ggaaagggca ggtgtgccgg
     2941 cgttccttga aacgtctgca ccacgtaacc ttccttttta cgagagattg gggtttacag
     3001 taacagcaga tgttgaagtg cctgaagggc cgaggacatg gtgcatgaca cgtaagccgg
     3061 gagcctaact cgagtgccca tttagttggc ttttcccttg tctcgtgtct tttccgtgga
     3121 aaggttcccg gagtaatctg atggcacagc agggaggtgc gcctgcaggt tggttaggaa
     3181 ggggggatga tgtaaaagaa gaaaatgggg ggattcgagc cccgcacagg tctctcaaat
     3241 tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta atcatggtca
     3301 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3361 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3421 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3481 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3541 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3601 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3661 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3721 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3781 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3841 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3901 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3961 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4021 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4081 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4141 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4201 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4261 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4321 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4381 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4441 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4501 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataattac gatacgggag
     4561 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4621 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4681 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4741 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4801 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4861 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4921 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4981 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5041 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5101 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5161 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5221 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5281 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5341 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5401 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga tgcggtgtga
     5461 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt
     5521 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     5581 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     5641 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     5701 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     5761 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     5821 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     5881 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc acttaatgcg
     5941 ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     6001 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     6061 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt
     6121 aatacgactc actata
//
