LOCUS       sequence-198947-        5718 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of sepHluorin fused to the cell penetrating 
            peptide TAT with a GST tag for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5718)
  AUTHORS   Richardson DS, Gregor C, Winter FR, Urban NT, Sahl SJ, Willig KI, 
            Hell SW
  TITLE     SRpHi ratiometric pH biosensors for super-resolution microscopy.
  JOURNAL   Nat Commun. 2017 Sep 18;8(1):577. doi: 10.1038/s41467-017-00606-4.
  PUBMED    28924139
REFERENCE   2  (bases 1 to 5718)
  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: "Nat 
            Commun."; date: "2017-09-18"; volume: "8(1):577. doi"; pages: " 
            10.1038/s41467-017-00606-4"
COMMENT     Sequence Label: SRpHi3
FEATURES             Location/Qualifiers
     source          1..5718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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)."
     CDS             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..759
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             808..1524
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(838..866)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1461..1481
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1586..1608)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1753..1771)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1839..1943
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1944..2012,2013..2804)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2162..2181)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2975..3563
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3464..3483
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3717..3734
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3807..3884
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3885..4967
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3904..3923)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5016..5021,5022..5039,5040..5046)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5054..5070
                     /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)."
     primer_bind     5078..5094
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5078..5094
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5090..5617
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5110..5127)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5110..5126)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5119..5141)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctgga agttctgttc caggggcccc tgggatccta tggccgaaag
      781 aagcggcgtc aacgacgtcg agaattcatg agtaaaggag aagaactttt cactggagtt
      841 gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtcagtgga
      901 gagggtgaag gtgatgcaac atacggaaaa cttaccctta aatttatttg cactactgga
      961 aaactacctg ttccttggcc aacacttgtc actactttaa cttatggtgt tcaatgcttt
     1021 tcaagatacc cagatcatat gaaacggcat gactttttca agagtgccat gcccgaaggt
     1081 tatgtacagg aaagaactat atttttcaaa gatgacggga actacaagac acgtgctgaa
     1141 gtcaagtttg aaggtgatac ccttgttaat agaatcgagt taaaaggtat tgattttaaa
     1201 gaagatggaa acattcttgg acacaaattg gaatacaact ataacgatca ccaggtgtac
     1261 atcatggcag acaaacaaaa gaatggaatc aaagctaact tcaaaattag acacaacatt
     1321 gaagatggag gcgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggg
     1381 cccgtccttt taccagacaa ccattacctg tttacaactt ctactctttc gaaagatccc
     1441 aacgaaaaga gagaccacat ggtccttctt gagtttgtaa cagctgctgg gattacacat
     1501 ggcatggatg aactatacaa ataaagcggc cgcatcgtga ctgactgacg atctgcctcg
     1561 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     1621 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     1681 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tataattctt
     1741 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     1801 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1861 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     1921 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     1981 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2041 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2101 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2161 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     2221 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2281 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2341 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2401 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     2461 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     2521 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     2581 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     2641 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     2701 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     2761 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     2821 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     2881 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2941 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3001 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3061 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3121 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3181 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3241 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3301 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3361 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3421 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3481 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3541 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3601 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3661 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3721 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3781 gcggtatttc acaccgcata aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta
     3841 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     3901 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     3961 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4021 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     4081 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     4141 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     4201 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     4261 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     4321 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     4381 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     4441 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     4501 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     4561 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     4621 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     4681 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     4741 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     4801 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     4861 gaaaaaccac gctagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     4921 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     4981 aatgtaagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     5041 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     5101 tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5161 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     5221 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgct ttgcctggtt
     5281 tccggcacca gaagcggtgc cggaaagctg gctggagtgc gatcttcctg aggccgatac
     5341 tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat gcgcccatct acaccaacgt
     5401 gacctatccc attacggtca atccgccgtt tgttcccacg gagaatccga cgggttgtta
     5461 ctcgctcaca tttaatgttg atgaaagctg gctacaggaa ggccagacgc gaattatttt
     5521 tgatggcgtt ggaattagct tatcgactgc acggtgcacc aatgcttctg gcgtcaggca
     5581 gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct
     5641 caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa
     5701 atattctgaa atgagctg
//
