LOCUS       sequence-355473-        6976 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 6976)
  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 6976)
  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-3mNG
FEATURES             Location/Qualifiers
     source          1..6976
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(4101..4119)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4102..4119)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4137..4153)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4137..4153)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4150..4172)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4161..4177
                     /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(4185..4191,4192..4209,4210..4215))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4230..4251
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4368..4385)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4539..5127)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4619..4638)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5298..6089,6090..6158))
                     /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     5921..5940
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6159..6263)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6341..6796)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6478..6499)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6690..6709
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6922..6944
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6936..6953
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6937..6953
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6960..6976,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6960..6976,1..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 tctcgaaagg tgaggaagac aatatggcca gcttgccagc aacgcacgag cttcatatat
     1021 ttgggtcgat caacggtgtg gatttcgaca tggtgggtca aggcacaggt aatcctaatg
     1081 acgggtatga agaactcaat ttgaagagca ctaagggaga tctccagttt tccccgtgga
     1141 ttcttgtgcc gcatattggt tatggttttc atcagtactt accatacccc gatggaatgt
     1201 cgcccttcca agcagctatg gtggacggct cggggtatca agtacatcgc accatgcaat
     1261 ttgaggacgg agcatccctc accgtaaact accgctatac gtatgagggg agccacataa
     1321 aaggtgaggc tcaagttaaa ggcacaggct tcccagctga cggcccggtc atgaccaatt
     1381 cattaaccgc tgctgattgg tgccgttcta agaagaccta ccctaacgac aagacgatca
     1441 ttagtacttt taagtggagt tataccacag gaaacggaaa gcggtatcga agcactgctc
     1501 gaacaaccta cacatttgcg aagccaatgg ccgcgaacta cttgaagaac cagccaatgt
     1561 atgtgtttcg aaaaacggaa ctgaagcatt cgaaaaccga gcttaatttt aaagagtggc
     1621 agaaggcgtt tacagacgtg atggggatgg acgaattgta taagcccggg ggcggctccg
     1681 gaagcgtgtc caagggtgag gaagataaca tggcctctct gcccgccacc cacgagttgc
     1741 atatctttgg aagcattaac ggagtggatt tcgatatggt gggtcagggg acaggaaatc
     1801 cgaacgatgg ttatgaagag ttgaacttga aaagcacgaa gggagacctc caattttcgc
     1861 cctggatctt ggtgccgcat atcggatatg ggttccatca atatttgccc taccctgacg
     1921 ggatgtcacc ctttcaggcc gcgatggttg atggttcggg ctatcaggta caccggacaa
     1981 tgcaatttga agatggtgcc tctttgacag tcaattaccg gtacacctat gaagggagcc
     2041 atattaaagg tgaagcccaa gtgaagggca ccggcttccc tgcggacggg cctgttatga
     2101 cgaactcact gactgcggca gattggtgta ggtcgaagaa gacttatcca aatgacaaaa
     2161 caataatctc cacatttaag tggtcgtaca caacaggaaa tggtaagcgt taccgctcga
     2221 ctgcgaggac gacctacaca ttcgccaagc ctatggcggc caactatctc aagaatcaac
     2281 ctatgtacgt gttccggaaa actgagttga aacactctaa aaccgaactc aacttcaagg
     2341 aatggcaaaa agcgtttacg gatgtaatgg gaatggatga attgtacaag ggctccggaa
     2401 gcggcagcgt atctaagggt gaagaggata atatggcgag cctgccagcc actcacgagc
     2461 ttcacatttt cgggtccata aatggcgtag actttgacat ggtaggacag gggacgggaa
     2521 accctaacga tggctatgag gagcttaact tgaaaagcac gaagggagat ctgcaattct
     2581 ctccgtggat tctcgttcca cacataggtt atgggtttca tcagtacctc ccctacccag
     2641 acggcatgag tccgttccaa gcagcaatgg tcgatggttc tggctatcaa gtccaccgta
     2701 ctatgcagtt tgaagatgga gcgagcctca cggtaaatta tcgttatacc tacgaagggt
     2761 ctcatattaa gggtgaagcc caggtgaagg ggaccggatt cccggccgat ggccctgtga
     2821 tgacgaatag ccttacggcc gctgactggt gtcggagtaa aaagacctat cccaatgaca
     2881 agacaattat ttccacgttt aaatggtcat atacgactgg gaatggtaag cgataccgat
     2941 ccactgcccg gacgacttat acctttgcta agcctatggc ggcgaactac ctcaagaatc
     3001 agccaatgta cgtgttccga aagacagagc tgaaacactc gaagactgaa ctcaatttta
     3061 aagaatggca aaaggccttc accgatgtga tgggaatgga tgaactgtat aaaggatccg
     3121 gatcaggatc tggatcagga tcgggtagtt aagagctccg ctgcgcttaa atgtctttca
     3181 ttataatcaa tgtataacct ttatgtagta tttcaatcta tgccgctgtg tacgtgcact
     3241 gcggtgccta tccttcggca ttagagagtc actgtttgtg tagatcgtag ctgcatgtct
     3301 gacatctggt tcattagtgc cgttttgagt ctgcactgtt gtgcaacttg atattcatca
     3361 ctgcacacca accgtgtgtt tcctcctttg ggtgcaccct ttaatccttg tcttctcctt
     3421 ttttgtctct ttccccctcg aaaaggtgtc aaactactgc cgcataaact acggttatcc
     3481 caaatttaag agaaagcaat aaagcatcag ctagcatgcc tttgtctcaa gaagaatcca
     3541 ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg
     3601 tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt
     3661 ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg
     3721 gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg
     3781 gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca
     3841 gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg
     3901 agggctaact cgagtgccca tttagttggc ttttcccttg tctcgtgtct tttccgtgga
     3961 aaggttcccg gagtaatctg atggcacagc agggaggtgc gcctgcaggt tggttaggaa
     4021 ggggggatga tgtaaaagaa gaaaatgggg ggattcgagc cccgcacagg tctctcaaat
     4081 tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta atcatggtca
     4141 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4201 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4261 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4321 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4381 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4441 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4501 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4561 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4621 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4681 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4741 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4801 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4861 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4921 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4981 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5041 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5101 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5161 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5221 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5281 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5341 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataattac gatacgggag
     5401 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5461 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5521 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5581 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5641 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5701 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5761 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5821 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5881 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5941 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6001 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6061 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6121 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6181 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6241 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga tgcggtgtga
     6301 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt
     6361 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     6421 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     6481 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     6541 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     6601 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     6661 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     6721 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc acttaatgcg
     6781 ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     6841 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     6901 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt
     6961 aatacgactc actata
//
