LOCUS       sequence-400069-        5554 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 5554)
  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 5554)
  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-blast-His:mScarletI:His
FEATURES             Location/Qualifiers
     source          1..5554
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             954..971
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1680..1697
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(2679..2697)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2680..2697)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2715..2731)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2715..2731)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2728..2750)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2739..2755
                     /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(2763..2769,2770..2787,2788..2793))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2808..2829
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2946..2963)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3117..3705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3197..3216)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3876..4667,4668..4736))
                     /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     4499..4518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4737..4841)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4919..5374)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5056..5077)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5268..5287
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5500..5522
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5514..5531
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5515..5531
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5538..5554,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5538..5554,1..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 tctcaccacc
      961 accaccatca caagcttatg gtatcaaagg gcgaagcagt aatcaaggag ttcatgcggt
     1021 ttaaggtaca catggagggt tccatgaatg gccatgaatt tgagatcgaa ggagaggggg
     1081 aagggcgtcc ttacgaagga acacaaacag ctaaacttaa ggtaactaag gggggacccc
     1141 tccccttttc ctgggacata ctgtcaccac aattcatgta cggcagccga gcctttataa
     1201 aacatccagc tgatataccc gattattata agcaaagttt tcccgagggc tttaagtggg
     1261 aaagggtcat gaacttcgag gacgggggtg cggtaactgt aacccaagac acctctctcg
     1321 aagacgggac gcttatttat aaggtgaaac tccgggggac caattttccc cctgacggac
     1381 ctgtcatgca gaagaagaca atgggctggg aggcgtccac agagcgcctg tatccggagg
     1441 atggcgtact taagggtgac ataaaaatgg cacttcgttt gaaggacggt ggccgttatc
     1501 tcgctgactt taagacaact tataaggcga aaaagccggt acagatgcca ggagcataca
     1561 atgttgaccg caaattggac atcaccagcc acaacgaaga ctataccgtc gttgaacagt
     1621 acgaacggag tgaagggagg cacagtacag gcgggatgga cgaattgtat aaaggatccc
     1681 accaccacca ccatcacgga tcaggatctg gatcaggatc gggtagttaa gagctccgct
     1741 gcgcttaaat gtctttcatt ataatcaatg tataaccttt atgtagtatt tcaatctatg
     1801 ccgctgtgta cgtgcactgc ggtgcctatc cttcggcatt agagagtcac tgtttgtgta
     1861 gatcgtagct gcatgtctga catctggttc attagtgccg ttttgagtct gcactgttgt
     1921 gcaacttgat attcatcact gcacaccaac cgtgtgtttc ctcctttggg tgcacccttt
     1981 aatccttgtc ttctcctttt ttgtctcttt ccccctcgaa aaggtgtcaa actactgccg
     2041 cataaactac ggttatccca aatttaagag aaagcaataa agcatcagct agcatgcctt
     2101 tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca
     2161 tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg
     2221 gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg
     2281 ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg
     2341 gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca
     2401 aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc
     2461 cctctggtta tgtgtgggag ggctaactcg agtgcccatt tagttggctt ttcccttgtc
     2521 tcgtgtcttt tccgtggaaa ggttcccgga gtaatctgat ggcacagcag ggaggtgcgc
     2581 ctgcaggttg gttaggaagg ggggatgatg taaaagaaga aaatgggggg attcgagccc
     2641 cgcacaggtc tctcaaattg gatgcatagc ttgagtattc tatagtgtca cctaaatagc
     2701 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     2761 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     2821 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     2881 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2941 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3001 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3061 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3121 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3181 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3241 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3301 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3361 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3421 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3481 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3541 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3601 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3661 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3721 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3781 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3841 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3901 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3961 ataattacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4021 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4081 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4141 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4201 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4261 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4321 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4381 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4441 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4501 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4561 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4621 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4681 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4741 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4801 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4861 ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa
     4921 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     4981 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     5041 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     5101 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     5161 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     5221 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     5281 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     5341 cccgccgcac ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact
     5401 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     5461 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     5521 cgacggccag tgaattgtaa tacgactcac tata
//
