LOCUS       sequence-170770-        3626 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of dual emitting pH sensitive GFP in bacterial 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3626)
  AUTHORS   Hanson GT, McAnaney TB, Park ES, Rendell ME, Yarbrough DK, Chu S, Xi
            L, Boxer SG, Montrose MH, Remington SJ
  TITLE     Green fluorescent protein variants as ratiometric dual emission pH 
            sensors. 1. Structural characterization and preliminary application.
  JOURNAL   Biochemistry. 2002 Dec 31;41(52):15477-88.
  PUBMED    12501176
REFERENCE   2  (bases 1 to 3626)
  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: 
            "Biochemistry."; date: "2002-12-31"; volume: "41(52)"; pages: 
            "15477-88"
COMMENT     Sequence Label: pRSETB-de4GFP
FEATURES             Location/Qualifiers
     source          1..3626
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             112..129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             133..165
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(169..177,178..192)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             211..924
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FISTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSCNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSCQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(241..269)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     864..884
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1027..1045)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1041..1088
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1185..1640
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1272..1291)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1482..1503
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1666..1770
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1771..1839,1840..2631)
                     /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(1989..2008)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2802..3390
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3291..3310
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3544..3561
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat
      121 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac
      181 gatgacgata aggatccccc cgctgaattc atgagtaaag gagaagaact tttcactgga
      241 gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt
      301 ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat ttccactact
      361 ggaaaactac ctgttccatg gccaacactt gtcactactt tcacttatgg tgttcaatgc
      421 ttttcaagat acccagatca tatgaaacgg catgactttt tcaagagtgc catgcccgaa
      481 ggttatgtac aggaaagaac tatatttttc aaagatgacg ggaactacaa gacacgtgct
      541 gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg agttaaaagg tattgatttt
      601 aaagaagatg gaaacattct tggacacaaa ttggaataca actataactc atgcaatgta
      661 tacatcatgg cagacaaaca aaagaatgga atcaaagtta acttcaaaat tagacacaac
      721 attgaagatg gaagcgttca actagcagac cattatcaac aaaatactcc aattggcgat
      781 ggccctgtcc ttttaccaga caaccattac ctgtcctgcc aatctgccct ttcgaaagat
      841 cccaacgaaa agagagacca catggtcctt cttgagtttg taacagctgc tgggattaca
      901 catggcatgg atgaactata caaataataa ggatccgagc tcgagatctg cagctggtac
      961 catggaattc gaagcttgat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1021 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1081 gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag aggcccgcac
     1141 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     1201 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1261 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1321 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1381 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1441 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1501 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1561 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1621 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1681 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     1741 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     1801 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     1861 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     1921 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     1981 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     2041 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     2101 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     2161 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     2221 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     2281 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     2341 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     2401 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     2461 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     2521 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     2581 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     2641 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     2701 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     2761 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2821 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2881 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2941 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3001 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3061 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3121 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3181 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3241 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3301 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3361 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3421 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3481 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     3541 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     3601 gcgcgttggc cgattcatta atgcag
//
