LOCUS       sequence-355418-        6145 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 6145)
  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 6145)
  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:mNG:3Ty
FEATURES             Location/Qualifiers
     source          1..6145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(3270..3288)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3271..3288)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3306..3322)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3306..3322)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3319..3341)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3330..3346
                     /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(3354..3360,3361..3378,3379..3384))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3399..3420
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3537..3554)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3708..4296)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3788..3807)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4467..5258,5259..5327))
                     /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     5090..5109
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5328..5432)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5510..5965)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5647..5668)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5859..5878
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6091..6113
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6105..6122
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6106..6122
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6129..6145,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6129..6145,1..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 ctcgaaaggt gaggaagaca atatggccag cttgccagca acgcacgagc
     1321 ttcatatatt tgggtcgatc aacggtgtgg atttcgacat ggtgggtcaa ggcacaggta
     1381 atcctaatga cgggtatgaa gaactcaatt tgaagagcac taagggagat ctccagtttt
     1441 ccccgtggat tcttgtgccg catattggtt atggttttca tcagtactta ccataccccg
     1501 atggaatgtc gcccttccaa gcagctatgg tggacggctc ggggtatcaa gtacatcgca
     1561 ccatgcaatt tgaggacgga gcatccctca ccgtaaacta ccgctatacg tatgagggga
     1621 gccacataaa aggtgaggct caagttaaag gcacaggctt cccagctgac ggcccggtca
     1681 tgaccaattc attaaccgct gctgattggt gccgttctaa gaagacctac cctaacgaca
     1741 agacgatcat tagtactttt aagtggagtt ataccacagg aaacggaaag cggtatcgaa
     1801 gcactgctcg aacaacctac acatttgcga agccaatggc cgcgaactac ttgaagaacc
     1861 agccaatgta tgtgtttcga aaaacggaac tgaagcattc gaaaaccgag cttaatttta
     1921 aagagtggca gaaggcgttt acagacgtga tggggatgga cgaattgtat aagggatccg
     1981 aggtacatac aaatcaggac cccctcgatg gtagtgaagt ccatacaaat caggacccgc
     2041 tggatggctc agaggttcat acgaaccaag atcctcttga tggatcagga tctggatcag
     2101 gatcgggtag ttaagagctc cgctgcgctt aaatgtcttt cattataatc aatgtataac
     2161 ctttatgtag tatttcaatc tatgccgctg tgtacgtgca ctgcggtgcc tatccttcgg
     2221 cattagagag tcactgtttg tgtagatcgt agctgcatgt ctgacatctg gttcattagt
     2281 gccgttttga gtctgcactg ttgtgcaact tgatattcat cactgcacac caaccgtgtg
     2341 tttcctcctt tgggtgcacc ctttaatcct tgtcttctcc ttttttgtct ctttccccct
     2401 cgaaaaggtg tcaaactact gccgcataaa ctacggttat cccaaattta agagaaagca
     2461 ataaagcatc agctagcatg actgaataca agccaacggt tcgtcttgct acgcgagacg
     2521 atgtgccgcg ggccgtacgg acgctggctg ctgcgttcgc tgattatccc gccacccgac
     2581 atacggttga cccagatcga cacattgagc gagtcacaga acttcaagag ttattcttaa
     2641 cacgggtggg cttggacatt ggtaaggttt gggttgcgga cgatggcgcg gctgttgcgg
     2701 tgtggacgac gccagagtca gtcgaagcgg gcgcagtgtt tgctgagatc ggtccccgaa
     2761 tggcggagct tagcgggtca cgcctcgctg cacaacagca gatggaaggt ctgttggcac
     2821 cacatagacc taaggagccg gcttggttct tagcgacagt tggcgtcagt ccggaccacc
     2881 aaggtaaggg gctgggttca gcggttgttc ttccaggagt agaggctgcg gaaagggcag
     2941 gtgtgccggc gttccttgaa acgtctgcac cacgtaacct tcctttttac gagagattgg
     3001 ggtttacagt aacagcagat gttgaagtgc ctgaagggcc gaggacatgg tgcatgacac
     3061 gtaagccggg agcctaactc gagtgcccat ttagttggct tttcccttgt ctcgtgtctt
     3121 ttccgtggaa aggttcccgg agtaatctga tggcacagca gggaggtgcg cctgcaggtt
     3181 ggttaggaag gggggatgat gtaaaagaag aaaatggggg gattcgagcc ccgcacaggt
     3241 ctctcaaatt ggatgcatag cttgagtatt ctatagtgtc acctaaatag cttggcgtaa
     3301 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3361 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3421 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3481 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3541 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3601 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3661 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3721 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3781 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3841 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3901 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3961 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4021 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4081 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4141 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4201 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4261 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4321 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4381 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4441 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4501 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataattacg
     4561 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4621 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4681 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4741 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4801 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4861 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4921 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4981 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5041 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5101 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5161 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5221 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5281 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5341 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5401 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgat
     5461 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     5521 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     5581 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     5641 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     5701 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     5761 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     5821 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     5881 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgca
     5941 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     6001 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     6061 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     6121 gtgaattgta atacgactca ctata
//
