LOCUS       sequence-423197-        6293 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the fluorescent protein pH sensor pHlorin2 in the 
            glycosomes of Trypanosomal brucei.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6293)
  TITLE     Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6293)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pLEW-pHluorin2-PTS1
FEATURES             Location/Qualifiers
     source          1..6293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(59..78)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(60..78)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    342..360
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    363..381
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(1986..2004)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1987..2004)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2294..2313)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(2523..2542)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2642..2664
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2702..2720)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2788..2892
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2893..2961,2962..3753)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3111..3130)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3924..4512
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4413..4432
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4666..4683
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(5839..6213)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
ORIGIN
        1 aaaaaaaaaa aaacatgaat ttcagaagac cttgctgtgc ccccggtacg ggagatctcc
       61 ctatagtgag tcgtattaat caggtaccgt cattggggtt aagcggaaag gtgtgtgtca
      121 gtaggttgtg aggtgaaagc gttttcagat gcatagtgag cttaatgtcc ttttcacagt
      181 atatcgtgtc tgataggtat ctcttattag tatagtcgaa tactagtcaa tagtgcgttt
      241 tgtgcaaaat gtccattttg tggcagtgat ggggttgttt tatgctattc cgtgtctctg
      301 ggtgggcgtg cattgaaaat aggggttatc gggtagggat ctccctatca gtgatagaga
      361 tctccctatc agtgatagag atccctgagt actgagttta acatgttctc gtcccgggct
      421 gcacgcgcct tcgagttttt tttccttttc cccatttttt tcaacttgaa gacttcaatt
      481 acaccaaaaa gtaaaattca caagcttgct agcatgagca aaggcgaaga attgtttacc
      541 ggtgttgtcc ctatcctggt cgagctggac ggcgatgtta acgggcataa gttcagtgtg
      601 tccggggaag gagaaggcga cgctacctac ggcaagctga cgctgaaatt catttgcacg
      661 acagggaagc tccccgtccc gtggcccacc cttgtcacga ccctgtccta tggggtacaa
      721 tgctttagtc gttatcccga tcacatgaaa caacatgact tctttaaaag tgcaatgccg
      781 gaaggttacg ttcaggagcg cacaatattc ttcaaggatg atggaaacta taagacaagg
      841 gctgaggtga aattcgaagg tgacactttg gtcaatcgaa ttgagctgaa agggattgat
      901 ttcaaggaag acggaaacat tctgggtcat aaactggaat acaactacaa tgaacatctt
      961 gtctacataa tggccgataa acaaaagaac gggactaaag ccatattcca agttcaccat
     1021 aacatcgaag atggcggcgt tcagctggcc gatcactacc aacaaaacac cccaatcggg
     1081 gatggtcctg tgcttctccc cgacaaccat taccttcata cgcaatccgc cttgagcaaa
     1141 gatcccaatg agaaaagaga tcatatggtt ctgctggagt tcgtgactgc tgcgggtatt
     1201 acgcacggca tggacgaact ttacaaaggc ggcgctaaac tgtaagaatt cggatcctgc
     1261 ccatttagtt ggcttttccc ttgtctcgtg tcttttccgt ggaaaggttc ccggagtaat
     1321 ctgatggcac agcagggagg tgcgcctgca ggttggttag gaagggggga tgatgtaaaa
     1381 gaagaaaatg gggggatatc ttatgtttaa gaggataaat aatgtgaagg ggctttatat
     1441 gcttgcttgg ttgtctcgtt ggtgcatggg gaatctgcat gtttgctttg gagcacgcgt
     1501 ggtacatcaa tgggatcata tcttgctgcc tcccgcagtc accgtgcgga gctgccggtg
     1561 ccccgtttct cataggcgtg tgcaccttcc gtctgagacc tgtaaatggt taaaaaggaa
     1621 tatataatta ccttttgaaa agtggtaaaa aacgaatata tgtttttttt tggttttaat
     1681 acgctctttt gtgtgtatga gaggaataaa taagtgtgtg tgtgtgtgtg tgttgttgtt
     1741 gttgaaaaaa tgaaggacac gaaagcgctc attaagggtg gggatacacc aggcataatt
     1801 atccgaaaat attttgatgc accaaataag tgaacatact gacgaaatca aagagttgga
     1861 ggatggatag ggaggcctca ttggcagtgg taaattgatt tgacttgaat atgataccct
     1921 cttactgttt cattgccact acaagttggt ttccttcccc tgcaggcatg caagctagct
     1981 tgtattctat agtgtcacct aaatcgtatg tgtatgatac ataaggttat gtattaattg
     2041 tagccgcgtt ctaacgacaa tatgtacaag cctaattgtg tagcatctgg cttactgaag
     2101 cagaccctat catctctctc gtaaactgcc gtcagagtcg gtttggttgg acgaaccttc
     2161 tgagtttctg gtaacgccgt cccgcacccg gaaatggtca gcgaaccaat cagcagggtc
     2221 atcgctagcc agatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
     2281 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc
     2341 gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg
     2401 ttgggcgcca tctccttgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
     2461 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgaatg gtgcactctc
     2521 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     2581 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     2641 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     2701 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     2761 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2821 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2881 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2941 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3001 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3061 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     3121 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     3181 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3241 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3301 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     3361 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     3421 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     3481 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     3541 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     3601 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     3661 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3721 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3781 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3841 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3901 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3961 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4021 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4081 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4141 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4201 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4261 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4321 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4381 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4441 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4501 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4561 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4621 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4681 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     4741 taatgcagaa ttcgagctca tatagttggt atgtattcta attccagact actggcgtgg
     4801 gataaacatg tccccctgat taaaaggaaa gattccatag cccattagtg caaagataat
     4861 tggtacaccg tcaaaaacac atgggagacc acacgatacg acactgtgac cgtagcatca
     4921 attccgcact cgatataact ctatcgaaga acttccatgg tacaaactgg tcgccacggt
     4981 ctgctccaca cggagatcat cgtatcattt ttatcgatag cggccgctat cgatgtatgc
     5041 cttggccctg atggcatgcc aatttcacta caacggacta tgtggacccc gttatcatgg
     5101 aaatgcgcta gttggaggaa gttagaccgc gccggaaaag agaggggtag agaaaatgac
     5161 aacttggaag atatccacac acgcacggtg aaacgttagc aacaattatt agggaagcga
     5221 cgcttgccct agtcccacga gtaaccaaga ctccaaaagc ctttctggca cagagagcga
     5281 gccgaaatgg aaaagagaaa caatgcctgc actaacacta ctgagcgatt cgcctcgccg
     5341 cggaggaccg aatactaata acgacacttg cggtcaaaaa gtagaagaac aaatgctcaa
     5401 cgatgagtga atcaggttag ggtagttgga aaattataca gaatgtcttt ggcaacacac
     5461 cgggcttgca tgcctgcaag gccttgcaag gccttgcaga atactgcata gataacaaac
     5521 gcatcaaaca caactggtgg cactatttca atcatgtcga cacaccaaga cgagaaaatt
     5581 tgagttaaac aagatgccta caaccctata tttattatct tcaagtgatc ttctcttctc
     5641 ttgtgcgctg tacgtaaatg tgttgcaaat aaggcatagg gctgagtaca ggcaccacac
     5701 gcgaggaaaa tcataagcac tagccgtgtc aactcacagc gactacagaa caattttggc
     5761 cacacaaccc ggtgttagga tctccgaggc ctcggacccg tcgggccgcg tcggaccggc
     5821 ggtgttggtc ggcgtcggtc agtcctgctc ctcggccacg aagtgcacgc agttgccggc
     5881 cgggtcgcgc agggcgaact cccgccccca cggctgctcg ccgatctcgg tcatggccgg
     5941 cccggaggcg tcccggaagt tcgtggacac gacctccgac cactcggcgt acagctcgtc
     6001 caggccgcgc acccacaccc aggccagggt gttgtccggc accacctggt cctggaccgc
     6061 gctgatgaac agggtcacgt cgtcccggac cacaccggcg aagtcgtcct ccacgaagtc
     6121 ccgggagaac ccgagccggt cggtccagaa ctcgaccgct ccggcgacgt cgcgcgcggt
     6181 gagcaccgga acggcactgg tcaacttggc catggtgata gcttatttta tggcagcaac
     6241 gagaccttac gtaaagcaaa actaaataag agcggagact gcaatgcaga gta
//
