LOCUS       sequence-194295-        4932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of fluorescent reporter for calcium signaling, 
            based off of GCaMP6s. Contains a stable reference Large Stokes Shift
            (LSS) mOrange nested within the reporting cpEGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4932)
  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 4932)
  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 MatryoshCaMP6s
FEATURES             Location/Qualifiers
     source          1..4932
                     /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     470..491
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             543..1250
                     /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     956..975
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1305..1326)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1566..1585)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2314..2332)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2328..2375
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2472..2927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2559..2578)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2769..2790
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2953..3057
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3058..3126,3127..3918)
                     /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(3276..3295)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4089..4677
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4578..4597
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4831..4848
                     /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 gagaacgtct atatcaaggc cgacaagcag aagaacggca
      301 tcaaggcgaa cttccacatc cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc
      361 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
      421 tgagcgtgca gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc
      481 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggta
      541 ccatggtgag caagggcgag gagaataaca tggccatcat caaggagttc atgcgcttca
      601 aggtgcgcat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
      661 gccgccccta cgagggcttt cagaccgtta agctgaaggt gaccaagggt ggccccctgc
      721 ccttcgcctg ggacatcttg tcccctcagt tcacctacgg ctccaaggcc tacgtgaagc
      781 accccgccga catccccgac tacctcaagc tgtccttccc cgagggcttc aagtgggagc
      841 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac tcaggactcc tccctgcagg
      901 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
      961 taatgcagaa gaagaccatg ggcatggagg cctcctccga gcggatgtac cccgaggacg
     1021 gcgccctgaa gggcgaggac aagctcaggc tgaagctgaa ggacggcggc cactacacct
     1081 ccgaggtcaa gaccacctac aaggccaaga agcccgtgca gttgcccggc gcctacatcg
     1141 tcgacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     1201 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag ggagggagca
     1261 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1321 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg tgagggcgat gccacctacg
     1381 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1441 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1501 agcacgactt cttcaagtcc gccatgcccg aaggctacat ccaggagcgc accatcttct
     1561 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1621 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1681 agctggagta caacctgccg gaccaactga ctgaagagca gatcgcagaa tttaaagagg
     1741 ctttctccct atttgacaag gacggggatg ggacaataac aaccaaggag ctggggacgg
     1801 tgatgcggtc tctggggcag aaccccacag aagcagagct gcaggacatg atcaatgaag
     1861 tagatgccga cggtgacggc acaatcgact tccctgagtt cctgacaatg atggcaagaa
     1921 aaatgaaata cagggacacg gaagaagaaa ttagagaagc gttcggtgtg tttgataagg
     1981 atggcaatgg ctacatcagt gcagcagagc ttcgccacgt gatgacaaac cttggagaga
     2041 agttaacaga tgaagaggtt gatgaaatga tcagggaagc agacatcgat ggggatggtc
     2101 aggtaaacta cgaagagttt gtacaaatga tgacagcgaa gtgagcggcc gcgactctag
     2161 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     2221 ctccccctga acctgaaaca taaaatgaat gcaattcgaa gcttgatccg gctgctaaca
     2281 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     2341 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     2401 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     2461 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2521 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2581 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2641 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2701 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2761 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2821 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2881 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     2941 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3001 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3061 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3121 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3181 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3241 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3301 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3361 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3421 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3481 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3541 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3601 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3661 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3721 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3781 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3841 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3901 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3961 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4021 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4081 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4141 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4201 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4261 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4321 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4381 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4441 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4501 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4561 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4621 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4681 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4741 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4801 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4861 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg caggatctcg
     4921 atcccgcgaa at
//
