LOCUS       sequence-441440-        5665 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 5665)
  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 5665)
  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: pPOTvGPI(VSG)-blast-mNG
FEATURES             Location/Qualifiers
     source          1..5665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2698..2716)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2699..2716)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2734..2750)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2734..2750)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2747..2769)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2758..2774
                     /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(2782..2788,2789..2806,2807..2812))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2827..2848
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2965..2982)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3136..3724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3216..3235)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3895..4686,4687..4755))
                     /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     4518..4537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4756..4860)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4938..5393)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5075..5096)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5287..5306
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5519..5541
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5533..5550
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5534..5550
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5557..5576
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5557..5575
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 attgcagtct ccgctcttat ttagttttgc tttacgtaag gtctcgttgc tgccataaaa
       61 taagctctag tggaattcat gcctttgtct caagaagaat ccaccctcat tgaaagagca
      121 acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc
      181 tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt
      241 gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc
      301 gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg
      361 cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg
      421 gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta actcgagtgc
      481 ccatttagtt ggcttttccc ttgtctcgtg tcttttccgt ggaaaggttc ccggagtaat
      541 ctgatggcac agcagggagg tgcgcctgca ggttggttag gaagggggga tgatgtaaaa
      601 gaagaaaatg gggggattcg agccccacgc gttgctcaag ctgtgtagcg caggcgtttc
      661 cttacatatt tctctaacag gcacggaagc ctaacaaata cacttggctt atttttttgc
      721 cccctcatgt cttgtacaaa tatttgcgat agcttagcta tcagccacat taatcaaaca
      781 agtataccaa caagcccgaa aacagaaact caactgcaac gggtacctat gcccagcaac
      841 caagaggcac gcctcttcct cgcagtgctc gtgctcgcac aagtgctccc cattctcgtg
      901 gacagcgcag cagagaaggg ttctggtagt ggttccggtt ccggttctaa gcttgtctcg
      961 aaaggtgagg aagacaatat ggccagcttg ccagcaacgc acgagcttca tatatttggg
     1021 tcgatcaacg gtgtggattt cgacatggtg ggtcaaggca caggtaatcc taatgacggg
     1081 tatgaagaac tcaatttgaa gagcactaag ggagatctcc agttttcccc gtggattctt
     1141 gtgccgcata ttggttatgg ttttcatcag tacttaccat accccgatgg aatgtcgccc
     1201 ttccaagcag ctatggtgga cggctcgggg tatcaagtac atcgcaccat gcaatttgag
     1261 gacggagcat ccctcaccgt aaactaccgc tatacgtatg aggggagcca cataaaaggt
     1321 gaggctcaag ttaaaggcac aggcttccca gctgacggcc cggtcatgac caattcatta
     1381 accgctgctg attggtgccg ttctaagaag acctacccta acgacaagac gatcattagt
     1441 acttttaagt ggagttatac cacaggaaac ggaaagcggt atcgaagcac tgctcgaaca
     1501 acctacacat ttgcgaagcc aatggccgcg aactacttga agaaccagcc aatgtatgtg
     1561 tttcgaaaaa cggaactgaa gcattcgaaa accgagctta attttaaaga gtggcagaag
     1621 gcgtttacag acgtgatggg gatggacgaa ttgtataagg gatccggatc aggatctgga
     1681 tcaggatcgg gtagtaacag cttcgtgatt agcaagacgc ccctctggct cgcagtgctc
     1741 ctcttctaag agctccgctg cgcttaaatg tctttcatta taatcaatgt ataaccttta
     1801 tgtagtattt caatctatgc cgctgtgtac gtgcactgcg gtgcctatcc ttcggcatta
     1861 gagagtcact gtttgtgtag atcgtagctg catgtctgac atctggttca ttagtgccgt
     1921 tttgagtctg cactgttgtg caacttgata ttcatcactg cacaccaacc gtgtgtttcc
     1981 tcctttgggt gcacccttta atccttgtct tctccttttt tgtctctttc cccctcgaaa
     2041 aggtgtcaaa ctactgccgc ataaactacg gttatcccaa atttaagaga aagcaataaa
     2101 gcatcagcta gcatgccttt gtctcaagaa gaatccaccc tcattgaaag agcaacggct
     2161 acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc tctctctagc
     2221 gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa
     2281 ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg tatcgtcgcg
     2341 atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca ggtgcttctc
     2401 gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc gacggcagtt
     2461 gggattcgtg aattgctgcc ctctggttat gtgtgggagg gctaactcga gtgcccattt
     2521 agttggcttt tcccttgtct cgtgtctttt ccgtggaaag gttcccggag taatctgatg
     2581 gcacagcagg gaggtgcgcc tgcaggttgg ttaggaaggg gggatgatgt aaaagaagaa
     2641 aatgggggga ttcgagcccc gcacaggtct ctcaaattgg atgcatagct tgagtattct
     2701 atagtgtcac ctaaatagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     2761 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     2821 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     2881 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     2941 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3001 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3061 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3121 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3181 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3241 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3301 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3361 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3421 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3481 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3541 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3601 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3661 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3721 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3781 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3841 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3901 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3961 ctgactcccc gtcgtgtaga taattacgat acgggagggc ttaccatctg gccccagtgc
     4021 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4081 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4141 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4201 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4261 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4321 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4381 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4441 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4501 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4561 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4621 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4681 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4741 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4801 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4861 cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat accgcacaga tgcgtaagga
     4921 gaaaataccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     4981 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     5041 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5101 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     5161 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     5221 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     5281 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     5341 gctgcgcgta accaccacac ccgccgcact taatgcgccg ctacagggcg cgtccattcg
     5401 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     5461 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     5521 cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa
     5581 ttgggcccgt ataatgcaga cctgctgcaa gctaggggca cagcaaggtc ttctgaaatt
     5641 catgtttttt ttttttttac tctgc
//
