LOCUS       sequence-400587-        6169 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 6169)
  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 6169)
  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:tagRFPt:3Ty
FEATURES             Location/Qualifiers
     source          1..6169
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(3294..3312)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3295..3312)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3330..3346)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3330..3346)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3343..3365)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3354..3370
                     /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(3378..3384,3385..3402,3403..3408))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3423..3444
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3561..3578)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3732..4320)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3812..3831)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4491..5282,5283..5351))
                     /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     5114..5133
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5352..5456)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5534..5989)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5671..5692)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5883..5902
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6115..6137
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6129..6146
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6130..6146
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6153..6169,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6153..6169,1..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 ataagcttgt ctcgaaggga gaggagctta taaaggaaaa tatgcacatg aagttatata
     1321 tggaaggaac ggttaataat caccatttta aatgtacgtc agaaggtgaa ggaaagccat
     1381 acgagggcac acagactatg aggataaagg ttgtcgaggg aggcccactg cctttcgcgt
     1441 tcgatattct cgccacgtct tttatgtatg gctcaaggac tttcatcaat catacgcagg
     1501 gcatcccgga ctttttcaag cagagttttc ctgaggggtt tacgtgggag cgtgttacca
     1561 catacgaaga tggaggagtg cttaccgcaa ctcaggacac ttcattacaa gacggatgcc
     1621 tgatctataa cgttaagatc cgcggagtga atttcccgag taacggacca gtgatgcaaa
     1681 agaaaacact tggatgggaa gctaacacgg agatgttgta tcccgcagat gggggtttgg
     1741 agggcaggac agacatggca ttaaaattgg tgggcggtgg tcacctgatc tgtaatttta
     1801 agacgacgta tcgaagtaag aaacctgcta agaacttgaa gatgccaggg gtctactacg
     1861 tcgatcatcg ccttgagagg attaaagagg cagataagga gacctatgta gaacagcacg
     1921 aggtggctgt ggcccgttac tgcgatctgc cctcgaagtt aggtcacaaa ttaaacggca
     1981 tggacgaatt gtacaaggga tccgaggtac atacaaatca ggaccccctc gatggtagtg
     2041 aagtccatac aaatcaggac ccgctggatg gctcagaggt tcatacgaac caagatcctc
     2101 ttgatggatc aggatctgga tcaggatcgg gtagttaaga gctccgctgc gcttaaatgt
     2161 ctttcattat aatcaatgta taacctttat gtagtatttc aatctatgcc gctgtgtacg
     2221 tgcactgcgg tgcctatcct tcggcattag agagtcactg tttgtgtaga tcgtagctgc
     2281 atgtctgaca tctggttcat tagtgccgtt ttgagtctgc actgttgtgc aacttgatat
     2341 tcatcactgc acaccaaccg tgtgtttcct cctttgggtg caccctttaa tccttgtctt
     2401 ctcctttttt gtctctttcc ccctcgaaaa ggtgtcaaac tactgccgca taaactacgg
     2461 ttatcccaaa tttaagagaa agcaataaag catcagctag catgactgaa tacaagccaa
     2521 cggttcgtct tgctacgcga gacgatgtgc cgcgggccgt acggacgctg gctgctgcgt
     2581 tcgctgatta tcccgccacc cgacatacgg ttgacccaga tcgacacatt gagcgagtca
     2641 cagaacttca agagttattc ttaacacggg tgggcttgga cattggtaag gtttgggttg
     2701 cggacgatgg cgcggctgtt gcggtgtgga cgacgccaga gtcagtcgaa gcgggcgcag
     2761 tgtttgctga gatcggtccc cgaatggcgg agcttagcgg gtcacgcctc gctgcacaac
     2821 agcagatgga aggtctgttg gcaccacata gacctaagga gccggcttgg ttcttagcga
     2881 cagttggcgt cagtccggac caccaaggta aggggctggg ttcagcggtt gttcttccag
     2941 gagtagaggc tgcggaaagg gcaggtgtgc cggcgttcct tgaaacgtct gcaccacgta
     3001 accttccttt ttacgagaga ttggggttta cagtaacagc agatgttgaa gtgcctgaag
     3061 ggccgaggac atggtgcatg acacgtaagc cgggagccta actcgagtgc ccatttagtt
     3121 ggcttttccc ttgtctcgtg tcttttccgt ggaaaggttc ccggagtaat ctgatggcac
     3181 agcagggagg tgcgcctgca ggttggttag gaagggggga tgatgtaaaa gaagaaaatg
     3241 gggggattcg agccccgcac aggtctctca aattggatgc atagcttgag tattctatag
     3301 tgtcacctaa atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3361 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3421 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3481 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3541 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3601 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3661 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3721 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3781 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3841 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3901 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3961 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4021 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4081 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4141 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4201 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4261 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4321 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4381 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4441 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4501 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4561 ctccccgtcg tgtagataat tacgatacgg gagggcttac catctggccc cagtgctgca
     4621 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4681 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4741 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4801 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4861 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4921 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4981 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     5041 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5101 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5161 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5221 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5281 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5341 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5401 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5461 tttccccgaa aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa
     5521 ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     5581 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     5641 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     5701 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     5761 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     5821 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     5881 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     5941 cgcgtaacca ccacacccgc cgcacttaat gcgccgctac agggcgcgtc cattcgccat
     6001 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     6061 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     6121 cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactata
//
