LOCUS       sequence-194294-        4926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of fluorescent reporter for calcium signaling, 
            based off of GCaMP6s. Contains a stable reference LSSmOrange nested 
            within the reporting superfolder cpGFP-T78H.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4926)
  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 4926)
  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 sfMatryoshCaMP6s-T78H
FEATURES             Location/Qualifiers
     source          1..4926
                     /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"
     CDS             540..1247
                     /codon_start=1
                     /product="monomeric orange fluorescent protein with a large
                     Stokes shift (Shcherbakova et al., 2012)"
                     /label=LSSmOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALK
                     GEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     953..972
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2308..2326)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2322..2369
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2466..2921
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2553..2572)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2763..2784
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2947..3051
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3052..3120,3121..3912)
                     /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(3270..3289)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4083..4671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4572..4591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4825..4842
                     /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 ggcggcacca
      541 tggtgagcaa gggcgaggag aataacatgg ccatcatcaa ggagttcatg cgcttcaagg
      601 tgcgcatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
      661 gcccctacga gggctttcag accgttaagc tgaaggtgac caagggtggc cccctgccct
      721 tcgcctggga catcttgtcc cctcagttca cctacggctc caaggcctac gtgaagcacc
      781 ccgccgacat ccccgactac ctcaagctgt ccttccccga gggcttcaag tgggagcgcg
      841 tgatgaactt cgaggacggc ggcgtggtga ccgtgactca ggactcctcc ctgcaggacg
      901 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
      961 tgcagaagaa gaccatgggc atggaggcct cctccgagcg gatgtacccc gaggacggcg
     1021 ccctgaaggg cgaggacaag ctcaggctga agctgaagga cggcggccac tacacctccg
     1081 aggtcaagac cacctacaag gccaagaagc ccgtgcagtt gcccggcgcc tacatcgtcg
     1141 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac
     1201 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagggc ggcagcgcga
     1261 gccagggcga agaactgttt accggcgtgg tgccgattct ggtggaactg gatggcgatg
     1321 tgaacggcca taaatttagc gtgcgcggcg aaggcgaagg cgatgcgacc attggcaaac
     1381 tgaccctgaa atttatttcc accaccggca aactaccggt gccgtggccg accctggtga
     1441 ccaccttaac ctatggcgtg cagtgcttta gccgctatcc ggatcatatg aaacgccatg
     1501 atttttttaa aagcgcgatg ccggaaggct atgtgcagga acgcaccatt agctttaaag
     1561 atgatggcaa atataaaacc cgcgcggtgg tgaaatttga aggcgatacc ctggtgaacc
     1621 gcattgaact gaaaggcacc gattttaaag aagatggcaa cattctgggg cataaactgg
     1681 aatataacct gccggaccaa ctgactgaag agcagatcgc agaatttaaa gaggctttct
     1741 ccctatttga caaggacggg gatgggacaa taacaaccaa ggagctgggg acggtgatgc
     1801 ggtctctggg gcagaacccc acagaagcag agctgcagga catgatcaat gaagtagatg
     1861 ccgacggtga cggcacaatc gacttccctg agttcctgac aatgatggca agaaaaatga
     1921 aatacaggga cacggaagaa gaaattagag aagcgttcgg tgtgtttgat aaggatggca
     1981 atggctacat cagtgcagca gagcttcgcc acgtgatgac aaaccttgga gagaagttaa
     2041 cagatgaaga ggttgatgaa atgatcaggg aagcagacat cgatggggat ggtcaggtaa
     2101 actacgaaga gtttgtacaa atgatgacag cgaagtgagc ggccgcgact ctagatcata
     2161 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     2221 ctgaacctga aacataaaat gaatgcaatt cgaagcttga tccggctgct aacaaagccc
     2281 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     2341 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatctggcg
     2401 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2461 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2521 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2581 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2641 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2701 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2761 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2821 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2881 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     2941 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3001 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3061 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3121 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3181 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3241 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     3301 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3361 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3421 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3481 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3541 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     3601 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3661 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3721 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3781 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3841 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3901 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     3961 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4021 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4081 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4141 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4201 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4261 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4321 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4381 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4441 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4501 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4561 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4621 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4681 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4741 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4801 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     4861 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcaggat ctcgatcccg
     4921 cgaaat
//
