LOCUS       sequence-400980-        6463 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 6463)
  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 6463)
  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-Ppyr9eh
FEATURES             Location/Qualifiers
     source          1..6463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(3588..3606)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3589..3606)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3624..3640)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3624..3640)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3637..3659)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3648..3664
                     /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(3672..3678,3679..3696,3697..3702))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3717..3738
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3855..3872)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4026..4614)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4106..4125)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4785..5576,5577..5645))
                     /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     5408..5427
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5646..5750)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5828..6283)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5965..5986)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6177..6196
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6409..6431
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6423..6440
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6424..6440
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6447..6463,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6447..6463,1..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 aggacgccaa gaacatcaag aagggaccag cccccttcta ccccctggag gacggcacag
     1021 ccggcgagca gctgcacaag gccatgaagc ggtacgccct ggtgccaggc accatcgcct
     1081 tcaccgacgc ccacatcgag gtgaacatca cctacgccga gtacttcgag atgagcgtgc
     1141 ggctggccga ggccatgaag cggtacggcc tgaacaccaa ccaccggatc gtggtgtgca
     1201 gcgagaacag cctgcagttc ttcatgcccg tgctgggagc cctgttcatc ggcgtggccg
     1261 tggccccagc caacgacatc tacaacgagc gggagctgct gaacagcatg aacatcagcc
     1321 agcccaccgt ggtgttcgtg agcaagaagg gcctgcagaa gatcctgaat gtgcagaaga
     1381 agctgcccat catccagaag atcatcatca tggacagcaa gaccgattac cagggcttcc
     1441 agagcatgta caccttcgtg accagccacc tgcccccagg cttcaacgag tacgacttcg
     1501 tgcccgagag cttcgaccgg gacaagacca tcgccctgat catgaacagc agcggcagca
     1561 ccggcctgcc caagggcgtg gccctgcccc accgggccct gtgcgtgcgg ttcagccacg
     1621 ccagagaccc catcttcggc aaccagatcg cccccgacac cgccatcctg agcgtggtgc
     1681 ccttccacca cggcttcggc atgttcacca ccctgggcta cctgatctgc ggcttccggg
     1741 tggtgctgat gtacaggttc gaggaggagc tgttcctgcg gagcctgcag gactacaaga
     1801 tccagaccgc cctgctggtg cccaccctgt tcagcttcct ggccaagagc accctgatcg
     1861 acaagtacga cctgagcaac ctgcacgaga tcgcctctgg cggagcccca ctgagcaagg
     1921 aggtgggcga ggccgtggcc aagggcttcc acctgccagg catccggcag ggctacggcc
     1981 tgaccgagac caccagcgcc atcctggtga cccccatcgg cgacgacaag cccggagccg
     2041 tgggcaaggt ggtgcccttc ttcgaggcca aggtggtgga cctggacacc ggcaagaccc
     2101 tgggcgtgaa ccagagaggc gagctgtgcg tgagaggccc catgatcatg agcggctacg
     2161 tgaacaaccc cgaggccacc aacgccctga tcgacaagga cggctggctg cacagcggcg
     2221 acatcgccta ctgggacgag gacgagcact tcttcatcgt ggaccggctg aagagcctga
     2281 tcaagtacaa gggctaccag gtggccccag ccgagctgga gagcatcctg ctgcagcacc
     2341 ccaacatccg ggacgccgga gtggccggac tgcccgacga cgacgccgga gagctgccag
     2401 ccgccgtggt ggtgctggag cacggcaaga ccatgaccga gaaggagatc gtggactacg
     2461 tggccagcca ggtgaccacc gccaagaagc tgagaggcgg cgtggtgttc gtggacgagg
     2521 tgcccaaggg cctgaccggc aagctggacg ccagaaagat ccgggagatc ctgatcaagg
     2581 ccaagaaggg cggcaagatc ggatccggat caggatctgg atcaggatcg ggtagttaag
     2641 agctccgctg cgcttaaatg tctttcatta taatcaatgt ataaccttta tgtagtattt
     2701 caatctatgc cgctgtgtac gtgcactgcg gtgcctatcc ttcggcatta gagagtcact
     2761 gtttgtgtag atcgtagctg catgtctgac atctggttca ttagtgccgt tttgagtctg
     2821 cactgttgtg caacttgata ttcatcactg cacaccaacc gtgtgtttcc tcctttgggt
     2881 gcacccttta atccttgtct tctccttttt tgtctctttc cccctcgaaa aggtgtcaaa
     2941 ctactgccgc ataaactacg gttatcccaa atttaagaga aagcaataaa gcatcagcta
     3001 gcatgccttt gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca
     3061 gcatccccat ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca
     3121 tcttcactgg tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc
     3181 tgggcactgc tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg
     3241 agaacagggg catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc
     3301 ctgggatcaa agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg
     3361 aattgctgcc ctctggttat gtgtgggagg gctaactcga gtgcccattt agttggcttt
     3421 tcccttgtct cgtgtctttt ccgtggaaag gttcccggag taatctgatg gcacagcagg
     3481 gaggtgcgcc tgcaggttgg ttaggaaggg gggatgatgt aaaagaagaa aatgggggga
     3541 ttcgagcccc gcacaggtct ctcaaattgg atgcatagct tgagtattct atagtgtcac
     3601 ctaaatagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3661 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3721 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3781 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3841 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3901 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3961 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4021 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4081 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4141 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4201 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4261 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4321 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4381 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4441 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4501 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4561 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4621 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4681 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4741 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4801 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4861 gtcgtgtaga taattacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4921 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4981 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5041 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5101 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5161 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5221 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5281 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5341 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5401 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5461 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5521 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5581 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5641 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5701 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5761 cgaaaagtgc cacctgatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     5821 catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     5881 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5941 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     6001 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     6061 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     6121 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     6181 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     6241 accaccacac ccgccgcact taatgcgccg ctacagggcg cgtccattcg ccattcaggc
     6301 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     6361 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     6421 gttgtaaaac gacggccagt gaattgtaat acgactcact ata
//
