LOCUS       sequence-194289-        7449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian 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 7449)
  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 7449)
  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: pcDNA3.2-DEST-MatryoshCaMP6s
FEATURES             Location/Qualifiers
     source          1..7449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    297..321
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             334..390
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     603..624
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             676..1383
                     /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     1089..1108
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1438..1459)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1699..1718)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(2275..2299)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2325..2366
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2406..2425)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2450..2498
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2700..3128
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2787..2806)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2997..3018
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3137..3157)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3142..3471
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3322..3457
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3384..3403
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3538..4332
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3592..3611)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4202..4221
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4508..4629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4545..4564)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4599..4618
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4678..4694)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4678..4694)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4691..4713)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4702..4718
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4726..4732,4733..4750,4751..4756))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4771..4792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4909..4926)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5080..5668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5160..5179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5839..6630,6631..6699))
                     /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     6462..6481
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6700..6804)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6879..6898)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7070..7449
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctg gctagttaag ctatcaacaa
      301 gtttgtacaa aaaagcagat ggtcgactca tcacgtcgta agtggaataa gacaggtcac
      361 gcagtcagag ctataggtcg gctgagctca ctcgagaacg tctatatcaa ggccgacaag
      421 cagaagaacg gcatcaaggc gaacttccac atccgccaca acatcgagga cggcggcgtg
      481 cagctcgcct accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
      541 gacaaccact acctgagcgt gcagtccaaa ctttcgaaag accccaacga gaagcgcgat
      601 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
      661 tacaagggcg gtaccatggt gagcaagggc gaggagaata acatggccat catcaaggag
      721 ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg gccacgagtt cgagatcgag
      781 ggcgagggcg agggccgccc ctacgagggc tttcagaccg ttaagctgaa ggtgaccaag
      841 ggtggccccc tgcccttcgc ctgggacatc ttgtcccctc agttcaccta cggctccaag
      901 gcctacgtga agcaccccgc cgacatcccc gactacctca agctgtcctt ccccgagggc
      961 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gactcaggac
     1021 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     1081 tccgacggcc ccgtaatgca gaagaagacc atgggcatgg aggcctcctc cgagcggatg
     1141 taccccgagg acggcgccct gaagggcgag gacaagctca ggctgaagct gaaggacggc
     1201 ggccactaca cctccgaggt caagaccacc tacaaggcca agaagcccgt gcagttgccc
     1261 ggcgcctaca tcgtcgacat caagttggac atcacctccc acaacgagga ctacaccatc
     1321 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     1381 aagggaggga gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1441 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggtgagggc
     1501 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1561 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1621 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta catccaggag
     1681 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1741 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1801 atcctggggc acaagctgga gtacaacctg ccggaccaac tgactgaaga gcagatcgca
     1861 gaatttaaag aggctttctc cctatttgac aaggacgggg atgggacaat aacaaccaag
     1921 gagctgggga cggtgatgcg gtctctgggg cagaacccca cagaagcaga gctgcaggac
     1981 atgatcaatg aagtagatgc cgacggtgac ggcacaatcg acttccctga gttcctgaca
     2041 atgatggcaa gaaaaatgaa atacagggac acggaagaag aaattagaga agcgttcggt
     2101 gtgtttgata aggatggcaa tggctacatc agtgcagcag agcttcgcca cgtgatgaca
     2161 aaccttggag agaagttaac agatgaagag gttgatgaaa tgatcaggga agcagacatc
     2221 gatggggatg gtcaggtaaa ctacgaagag tttgtacaaa tgatgacagc gaagtgacag
     2281 ctttcttgta caaagtggtt gatctagagg gcccgcggtt cgaaggtaag cctatcccta
     2341 accctctcct cggtctcgat tctacgcgta ccggttagta atgagtttaa acgggggagg
     2401 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     2461 agacagaata aaacgcacgg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     2521 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     2581 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     2641 cggggcggca ggccctgcca tagcagatct gcgcagctgg ggctctaggg ggtatcccca
     2701 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2761 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2821 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2881 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2941 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3001 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3061 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3121 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3181 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3241 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3301 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3361 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     3421 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     3481 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     3541 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3601 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3661 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     3721 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     3781 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     3841 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     3901 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     3961 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4021 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     4081 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4141 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4201 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4261 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4321 gagttcttct gagcgggact ctggggttcg cgaaatgacc gaccaagcga cgcccaacct
     4381 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4441 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4501 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4561 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4621 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     4681 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4741 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4801 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4861 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4921 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4981 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5041 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5101 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5161 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5221 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5281 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5341 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5401 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5461 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5521 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5581 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5641 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5701 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5761 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5821 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5881 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5941 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6001 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6061 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6121 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6181 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6241 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6301 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6361 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6421 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6481 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6541 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6601 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     6661 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     6721 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     6781 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     6841 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
     6901 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
     6961 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
     7021 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
     7081 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     7141 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     7201 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     7261 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     7321 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     7381 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     7441 attaccatg
//
