LOCUS       sequence-325096-        3327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for: ratiometric pHluorin (endoplasmic reticulum).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3327)
  AUTHORS   Werley CA, Boccardo S, Rigamonti A, Hansson EM, Cohen AE
  TITLE     Multiplexed Optical Sensors in Arrayed Islands of Cells for 
            multimodal recordings of cellular physiology.
  JOURNAL   Nat Commun. 2020 Aug 4;11(1):3881. doi: 10.1038/s41467-020-17607-5.
  PUBMED    32753572
REFERENCE   2  (bases 1 to 3327)
  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: "2020-08-4"; volume: "11(1):3881. doi"; pages: " 
            10.1038/s41467-020-17607-5"
COMMENT     Sequence Label: pMOS030E: Entry vector for: ratiometric pHluor...
FEATURES             Location/Qualifiers
     source          1..3327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             723..1436
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Miesenbock et al., 1998)"
                     /label=ratiometric pHluorin
                     /note="responds to a drop in pH with a reversible decrease 
                     in excitation at 395 nm and a corresponding increase in 
                     excitation at 475 nm"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKDDGNILGHKLEYNYNEHLVYIMADKQKNGTKA
                     IFQVHHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLHTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(753..781)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1376..1396
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    complement(1452..1551)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1568..1587)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1569..1587)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1592..1608)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1592..1608)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1710..1729)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1721..2530
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1788..1807)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2677..3265
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3166..3185
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctac catgctgcta tccgtgccgt tgctgctcgg cctcctcggc ctggccgctg
      721 caatgagtaa aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg
      781 gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg
      841 gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac
      901 ttgtcactac tttctcttat ggtgttcaat gcttttcaag atacccagat catatgaaac
      961 ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt
     1021 tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg
     1081 ttaatagaat cgagttaaaa ggtattgatt ttaaagatga tggaaacatt cttggacaca
     1141 aattggaata caactataac gagcacttgg tgtacatcat ggcagacaaa caaaagaatg
     1201 gtaccaaagc tatctttcaa gttcaccaca acattgaaga tggaggcgtt caactagcag
     1261 accattatca acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt
     1321 acctgcacac acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc
     1381 ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaaaagg
     1441 acgagctgta aacccagctt tcttgtacaa agttggcatt ataagaaagc attgcttatc
     1501 aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc
     1561 tgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc
     1621 cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat
     1681 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     1741 aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     1801 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
     1861 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     1921 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     1981 tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt
     2041 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     2101 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     2161 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     2221 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     2281 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     2341 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     2401 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     2461 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     2521 gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg
     2581 acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
     2641 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2701 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2761 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2821 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2881 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2941 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3001 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3061 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3121 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3181 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3241 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3301 cttttgctgg ccttttgctc acatgtt
//
