LOCUS       sequence-403333-        5518 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 5518)
  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 5518)
  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-mScarletI
FEATURES             Location/Qualifiers
     source          1..5518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     437..456
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(675..779)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(857..1312)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(994..1015)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1206..1225
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1438..1460
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1452..1469
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1453..1469
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1476..1495
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1476..1494
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4135..4153)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4136..4153)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4171..4187)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4171..4187)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4184..4206)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4195..4211
                     /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(4219..4225,4226..4243,4244..4249))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4264..4285
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4402..4419)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4573..5161)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4653..4672)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5332..5518,1..605,606..674))
                     /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"
ORIGIN
        1 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
       61 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
      121 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
      181 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
      241 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
      301 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
      361 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
      421 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
      481 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
      541 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
      601 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
      661 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
      721 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
      781 acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag
      841 aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
      901 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
      961 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     1021 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     1081 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     1141 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     1201 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     1261 ctgcgcgtaa ccaccacacc cgccgcactt aatgcgccgc tacagggcgc gtccattcgc
     1321 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     1381 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     1441 agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat
     1501 tgggcccgta taatgcagac ctgctgcaag ctaggggcac agcaaggtct tctgaaattc
     1561 atgttttttt tttttttact ctgcattgca gtctccgctc ttatttagtt ttgctttacg
     1621 taaggtctcg ttgctgccat aaaataagct ctagtggaat tcatgccttt gtctcaagaa
     1681 gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac
     1741 tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta
     1801 tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg
     1861 gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc
     1921 ccctgcggac ggtgccgaca ggtgcttctc gacctgcatc ctgggatcaa agccatagtg
     1981 aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat
     2041 gtgtgggagg gctaaccatg gtgcccattt agttggcctt tcccttgtct cgtgtctttt
     2101 ccgtggaaag gttcccggag taatctgatg gcacagcagg gaggtgcgcc tgcaggttgg
     2161 ttaggaaggg gggatgatgt aaaagaagaa aatgggggga ttcgagcccc acgcgttgct
     2221 caagctgtgt agcgcaggcg tttccttaca tatttctcta acaggcacgg aagcctaaca
     2281 aatacacttg gcttattttt ttgccccctc atgtcttgta caaatatttg cgatagctta
     2341 gctatcagcc acattaatca aacaagtata ccaacaagcc cgaaaacaga aactcaactg
     2401 caacgggtac ctatgggttc tggtagtggt tccggttccg gttctaagct tatggtatca
     2461 aagggcgaag cagtaatcaa ggagttcatg cggtttaagg tacacatgga gggttccatg
     2521 aatggccatg aatttgagat cgaaggagag ggggaagggc gtccttacga aggaacacaa
     2581 acagctaaac ttaaggtaac taagggggga cccctcccct tttcctggga catactgtca
     2641 ccacaattca tgtacggcag ccgagccttt ataaaacatc cagctgatat acccgattat
     2701 tataagcaaa gttttcccga gggctttaag tgggaaaggg tcatgaactt cgaggacggg
     2761 ggtgcggtaa ctgtaaccca agacacctct ctcgaagacg ggacgcttat ttataaggtg
     2821 aaactccggg ggaccaattt tccccctgac ggacctgtca tgcagaagaa gacaatgggc
     2881 tgggaggcgt ccacagagcg cctgtatccg gaggatggcg tacttaaggg tgacataaaa
     2941 atggcacttc gtttgaagga cggtggccgt tatctcgctg actttaagac aacttataag
     3001 gcgaaaaagc cggtacagat gccaggagca tacaatgttg accgcaaatt ggacatcacc
     3061 agccacaacg aagactatac cgtcgttgaa cagtacgaac ggagtgaagg gaggcacagt
     3121 acaggcggga tggacgaatt gtataaagga tccggatcag gatctggatc aggatcgggt
     3181 agttaagagc tccgctgcgc ttaaatgtct ttcattataa tcaatgtata acctttatgt
     3241 agtatttcaa tctatgccgc tgtgtacgtg cactgcggtg cctatccttc ggcattagag
     3301 agtcactgtt tgtgtagatc gtagctgcat gtctgacatc tggttcatta gtgccgtttt
     3361 gagtctgcac tgttgtgcaa cttgatattc atcactgcac accaaccgtg tgtttcctcc
     3421 tttgggtgca ccctttaatc cttgtcttct ccttttttgt ctctttcccc ctcgaaaagg
     3481 tgtcaaacta ctgccgcata aactacggtt atcccaaatt taagagaaag caataaagca
     3541 tcagctagca tgcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca
     3601 atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac
     3661 ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc
     3721 gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc
     3781 ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat
     3841 ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg
     3901 attcgtgaat tgctgccctc tggttatgtg tgggagggct aactcgagtg cccatttagt
     3961 tggcttttcc cttgtctcgt gtcttttccg tggaaaggtt cccggagtaa tctgatggca
     4021 cagcagggag gtgcgcctgc aggttggtta ggaagggggg atgatgtaaa agaagaaaat
     4081 ggggggattc gagccccgca caggtctctc aaattggatg catagcttga gtattctata
     4141 gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4201 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4261 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4321 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4381 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4441 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4501 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4561 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4621 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4681 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4741 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4801 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4861 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4921 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4981 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5041 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5101 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5161 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5221 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5281 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5341 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5401 actccccgtc gtgtagataa ttacgatacg ggagggctta ccatctggcc ccagtgctgc
     5461 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagcca
//
