LOCUS       sequence-194290-        4218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of fluorescent reporter for calcium signaling, 
            based off of GCaMP6s. cpEGFP replaced with a superfolder cpGFP 
            variant containing T78H mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4218)
  AUTHORS   Ast C, Foret J, Oltrogge LM, De Michele R, Kleist TJ, Ho CH, Frommer
            WB
  TITLE     Ratiometric Matryoshka biosensors from a nested cassette of green- 
            and orange-emitting fluorescent proteins.
  JOURNAL   Nat Commun. 2017 Sep 5;8(1):431. doi: 10.1038/s41467-017-00400-2.
  PUBMED    28874729
REFERENCE   2  (bases 1 to 4218)
  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: "2017-09-5"; volume: "8(1):431. doi"; pages: " 
            10.1038/s41467-017-00400-2"
COMMENT     Sequence Label: pRSET a sfGCaMP6s-T78H
FEATURES             Location/Qualifiers
     source          1..4218
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             93..110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             114..146
                     /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(150..158,159..173)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      180..189
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             201..257
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     complement(1600..1618)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1614..1661
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1758..2213
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1845..1864)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2055..2076
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2239..2343
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2344..2412,2413..3204)
                     /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(2562..2581)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3375..3963
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3864..3883
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4117..4134
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacat atgcggggtt ctcatcatca tcatcatcat ggtatggcta
      121 gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatctcg
      181 ccaccatggt cgactcatca cgtcgtaagt ggaataagac aggtcacgca gtcagagcta
      241 taggtcggct gagctcactc gagaacgtgt atattaccgc ggataaacag aaaaacggca
      301 ttaaagcgaa ctttcacgtg cgccataacg tggaagatgg cagcgtgcag ctggcggatc
      361 attatcagca gaacaccccg attggcgatg gcccggtgct gctgccggat aaccattatc
      421 tgagcaccca gaccaagctg agcaaagatc cgaacgaaaa acgcgatcac atggtgctgc
      481 tggaatttgt gaccgcagcg ggcattacac acggcatgga tgaactgtat ggcggcaccg
      541 gcggcagcgc gagccagggc gaagaactgt ttaccggcgt ggtgccgatt ctggtggaac
      601 tggatggcga tgtgaacggc cataaattta gcgtgcgcgg cgaaggcgaa ggcgatgcga
      661 ccattggcaa actgaccctg aaatttattt ccaccaccgg caaactaccg gtgccgtggc
      721 cgaccctggt gaccacctta acctatggcg tgcagtgctt tagccgctat ccggatcata
      781 tgaaacgcca tgattttttt aaaagcgcga tgccggaagg ctatgtgcag gaacgcacca
      841 ttagctttaa agatgatggc aaatataaaa cccgcgcggt ggtgaaattt gaaggcgata
      901 ccctggtgaa ccgcattgaa ctgaaaggca ccgattttaa agaagatggc aacattctgg
      961 ggcataaact ggaatataac ctgccggacc aactgactga agagcagatc gcagaattta
     1021 aagaggcttt ctccctattt gacaaggacg gggatgggac aataacaacc aaggagctgg
     1081 ggacggtgat gcggtctctg gggcagaacc ccacagaagc agagctgcag gacatgatca
     1141 atgaagtaga tgccgacggt gacggcacaa tcgacttccc tgagttcctg acaatgatgg
     1201 caagaaaaat gaaatacagg gacacggaag aagaaattag agaagcgttc ggtgtgtttg
     1261 ataaggatgg caatggctac atcagtgcag cagagcttcg ccacgtgatg acaaaccttg
     1321 gagagaagtt aacagatgaa gaggttgatg aaatgatcag ggaagcagac atcgatgggg
     1381 atggtcaggt aaactacgaa gagtttgtac aaatgatgac agcgaagtga gcggccgcga
     1441 ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     1501 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttcgaagctt gatccggctg
     1561 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     1621 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1681 ccggatctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     1741 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1801 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1861 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1921 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1981 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2041 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2101 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2161 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     2221 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2281 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2341 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2401 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2461 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2521 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2581 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2641 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     2701 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     2761 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     2821 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     2881 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     2941 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3001 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3061 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3121 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3181 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3241 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3301 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3361 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3421 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3481 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     3541 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3601 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3661 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3721 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3781 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3841 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3901 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3961 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4021 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4081 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4141 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagg
     4201 atctcgatcc cgcgaaat
//
