LOCUS       sequence-400978-        7174 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 7174)
  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 7174)
  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-mNG:Ppyr9eh
FEATURES             Location/Qualifiers
     source          1..7174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(4299..4317)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4300..4317)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4335..4351)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4335..4351)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4348..4370)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4359..4375
                     /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(4383..4389,4390..4407,4408..4413))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4428..4449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4566..4583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4737..5325)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4817..4836)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5496..6287,6288..6356))
                     /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     6119..6138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6357..6461)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6539..6994)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6676..6697)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6888..6907
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7120..7142
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7134..7151
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7135..7151
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(7158..7174,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(7158..7174,1..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 gaggacgcca
     1681 agaacatcaa gaagggacca gcccccttct accccctgga ggacggcaca gccggcgagc
     1741 agctgcacaa ggccatgaag cggtacgccc tggtgccagg caccatcgcc ttcaccgacg
     1801 cccacatcga ggtgaacatc acctacgccg agtacttcga gatgagcgtg cggctggccg
     1861 aggccatgaa gcggtacggc ctgaacacca accaccggat cgtggtgtgc agcgagaaca
     1921 gcctgcagtt cttcatgccc gtgctgggag ccctgttcat cggcgtggcc gtggccccag
     1981 ccaacgacat ctacaacgag cgggagctgc tgaacagcat gaacatcagc cagcccaccg
     2041 tggtgttcgt gagcaagaag ggcctgcaga agatcctgaa tgtgcagaag aagctgccca
     2101 tcatccagaa gatcatcatc atggacagca agaccgatta ccagggcttc cagagcatgt
     2161 acaccttcgt gaccagccac ctgcccccag gcttcaacga gtacgacttc gtgcccgaga
     2221 gcttcgaccg ggacaagacc atcgccctga tcatgaacag cagcggcagc accggcctgc
     2281 ccaagggcgt ggccctgccc caccgggccc tgtgcgtgcg gttcagccac gccagagacc
     2341 ccatcttcgg caaccagatc gcccccgaca ccgccatcct gagcgtggtg cccttccacc
     2401 acggcttcgg catgttcacc accctgggct acctgatctg cggcttccgg gtggtgctga
     2461 tgtacaggtt cgaggaggag ctgttcctgc ggagcctgca ggactacaag atccagaccg
     2521 ccctgctggt gcccaccctg ttcagcttcc tggccaagag caccctgatc gacaagtacg
     2581 acctgagcaa cctgcacgag atcgcctctg gcggagcccc actgagcaag gaggtgggcg
     2641 aggccgtggc caagggcttc cacctgccag gcatccggca gggctacggc ctgaccgaga
     2701 ccaccagcgc catcctggtg acccccatcg gcgacgacaa gcccggagcc gtgggcaagg
     2761 tggtgccctt cttcgaggcc aaggtggtgg acctggacac cggcaagacc ctgggcgtga
     2821 accagagagg cgagctgtgc gtgagaggcc ccatgatcat gagcggctac gtgaacaacc
     2881 ccgaggccac caacgccctg atcgacaagg acggctggct gcacagcggc gacatcgcct
     2941 actgggacga ggacgagcac ttcttcatcg tggaccggct gaagagcctg atcaagtaca
     3001 agggctacca ggtggcccca gccgagctgg agagcatcct gctgcagcac cccaacatcc
     3061 gggacgccgg agtggccgga ctgcccgacg acgacgccgg agagctgcca gccgccgtgg
     3121 tggtgctgga gcacggcaag accatgaccg agaaggagat cgtggactac gtggccagcc
     3181 aggtgaccac cgccaagaag ctgagaggcg gcgtggtgtt cgtggacgag gtgcccaagg
     3241 gcctgaccgg caagctggac gccagaaaga tccgggagat cctgatcaag gccaagaagg
     3301 gcggcaagat cggatccgga tcaggatctg gatcaggatc gggtagttaa gagctccgct
     3361 gcgcttaaat gtctttcatt ataatcaatg tataaccttt atgtagtatt tcaatctatg
     3421 ccgctgtgta cgtgcactgc ggtgcctatc cttcggcatt agagagtcac tgtttgtgta
     3481 gatcgtagct gcatgtctga catctggttc attagtgccg ttttgagtct gcactgttgt
     3541 gcaacttgat attcatcact gcacaccaac cgtgtgtttc ctcctttggg tgcacccttt
     3601 aatccttgtc ttctcctttt ttgtctcttt ccccctcgaa aaggtgtcaa actactgccg
     3661 cataaactac ggttatccca aatttaagag aaagcaataa agcatcagct agcatgcctt
     3721 tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca
     3781 tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg
     3841 gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg
     3901 ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg
     3961 gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca
     4021 aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc
     4081 cctctggtta tgtgtgggag ggctaactcg agtgcccatt tagttggctt ttcccttgtc
     4141 tcgtgtcttt tccgtggaaa ggttcccgga gtaatctgat ggcacagcag ggaggtgcgc
     4201 ctgcaggttg gttaggaagg ggggatgatg taaaagaaga aaatgggggg attcgagccc
     4261 cgcacaggtc tctcaaattg gatgcatagc ttgagtattc tatagtgtca cctaaatagc
     4321 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4381 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4441 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4501 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4561 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4621 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4681 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4741 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4801 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4861 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4921 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4981 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5041 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5101 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5161 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5221 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5281 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5341 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5401 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5461 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5521 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5581 ataattacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5641 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5701 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5761 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5821 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5881 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5941 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6001 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6061 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6121 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6181 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6241 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6301 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6361 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6421 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6481 ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa
     6541 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     6601 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     6661 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     6721 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     6781 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     6841 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     6901 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     6961 cccgccgcac ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact
     7021 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     7081 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     7141 cgacggccag tgaattgtaa tacgactcac tata
//
