LOCUS       sequence-182904-        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..
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
FEATURES             Location/Qualifiers
     source          1..4218
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             112..129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             133..165
                     /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(169..177,178..192)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      199..208
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             220..276
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     complement(1619..1637)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1633..1680
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1777..2232
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1864..1883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2074..2095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2258..2362
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2363..2431,2432..3223)
                     /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(2581..2600)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3394..3982
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3883..3902
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4136..4153
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat
      121 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac
      181 gatgacgata aggatctcgc caccatggtc gactcatcac gtcgtaagtg gaataagaca
      241 ggtcacgcag tcagagctat aggtcggctg agctcactcg agaacgtgta tattaccgcg
      301 gataaacaga aaaacggcat taaagcgaac tttaccgtgc gccataacgt ggaagatggc
      361 agcgtgcagc tggcggatca ttatcagcag aacaccccga ttggcgatgg cccggtgctg
      421 ctgccggata accattatct gagcacccag accaagctga gcaaagatcc gaacgaaaaa
      481 cgcgatcaca tggtgctgct ggaatttgtg accgcagcgg gcattacaca cggcatggat
      541 gaactgtatg gcggcaccgg cggcagcgcg agccagggcg aagaactgtt taccggcgtg
      601 gtgccgattc tggtggaact ggatggcgat gtgaacggcc ataaatttag cgtgcgcggc
      661 gaaggcgaag gcgatgcgac cattggcaaa ctgaccctga aatttatttc caccaccggc
      721 aaactaccgg tgccgtggcc gaccctggtg accaccttaa cctatggcgt gcagtgcttt
      781 agccgctatc cggatcatat gaaacgccat gattttttta aaagcgcgat gccggaaggc
      841 tatgtgcagg aacgcaccat tagctttaaa gatgatggca aatataaaac ccgcgcggtg
      901 gtgaaatttg aaggcgatac cctggtgaac cgcattgaac tgaaaggcac cgattttaaa
      961 gaagatggca acattctggg gcataaactg gaatataacc tgccggacca actgactgaa
     1021 gagcagatcg cagaatttaa agaggctttc tccctatttg acaaggacgg ggatgggaca
     1081 ataacaacca aggagctggg gacggtgatg cggtctctgg ggcagaaccc cacagaagca
     1141 gagctgcagg acatgatcaa tgaagtagat gccgacggtg acggcacaat cgacttccct
     1201 gagttcctga caatgatggc aagaaaaatg aaatacaggg acacggaaga agaaattaga
     1261 gaagcgttcg gtgtgtttga taaggatggc aatggctaca tcagtgcagc agagcttcgc
     1321 cacgtgatga caaaccttgg agagaagtta acagatgaag aggttgatga aatgatcagg
     1381 gaagcagaca tcgatgggga tggtcaggta aactacgaag agtttgtaca aatgatgaca
     1441 gcgaagtgag cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
     1501 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     1561 tcgaagcttg atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1621 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1681 ctgaaaggag gaactatatc cggatctggc gtaatagcga agaggcccgc accgatcgcc
     1741 cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa
     1801 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1861 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1921 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1981 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2041 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2101 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2161 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2221 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2281 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     2341 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     2401 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     2461 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     2521 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     2581 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     2641 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     2701 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     2761 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     2821 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     2881 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     2941 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3001 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3061 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     3121 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     3181 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     3241 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     3301 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3361 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3421 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3481 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3541 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3601 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3661 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3721 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3781 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3841 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3901 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3961 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4021 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4081 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4141 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     4201 gccgattcat taatgcag
//
