LOCUS       sequence-69435-e        4543 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for calcium signaling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4543)
  AUTHORS   Thestrup T, Litzlbauer J, Bartholomaus I, Mues M, Russo L, Dana H, 
            Kovalchuk Y, Liang Y, Kalamakis G, Laukat Y, Becker S, Witte G, 
            Geiger A, Allen T, Rome LC, Chen TW, Kim DS, Garaschuk O, Griesinger
            C, Griesbeck O
  TITLE     Optimized ratiometric calcium sensors for functional in vivo imaging
            of neurons and T lymphocytes.
  JOURNAL   Nat Methods. 2014 Jan 5. doi: 10.1038/nmeth.2773.
  PUBMED    24390440
REFERENCE   2  (bases 1 to 4543)
  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 
            Methods. 2014 Jan 5. doi: 10.1038/nmeth.2773."
COMMENT     Sequence Label: Twitch-2B pRSETB
FEATURES             Location/Qualifiers
     source          1..4543
                     /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      205..214
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(256..277)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(517..536)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(1944..1962)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1958..2005
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2102..2557
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2189..2208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2399..2420
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2583..2687
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2688..2756,2757..3548)
                     /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(2906..2925)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3719..4307
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4208..4227
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4461..4478
                     /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 aggatcccgc ggccgccacc atggtgagca agggcgagga gctgttcacc
      241 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      301 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      361 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgagctg gggcgtgcag
      421 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
      481 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
      541 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
      601 ttcaaggagg acggcaacat cctggggcac aagctggagt acaacgccat ccacggcaac
      661 gtctatatca ccgccgacaa gcagaagaac ggcatcaagg ccaacttcgg cctcaactgc
      721 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
      781 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa gctgagcaaa
      841 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgcccgcatg
      901 caagttgccg acgccagcga agaggaactg agcgagtgct tcagaatctt cgacttcgac
      961 ggcaacggct tcatcgacag agaggagttt ggcgacatca tcagactgac cggcgagcag
     1021 ctgaccgacg aggacgtgga cgagatcttc ggcgactccg acaccgacaa gaacggcaga
     1081 atcgatttcg acgagttcct gaagatggtg gaaaacgtgc agcccatcta ccccgagctc
     1141 atgggtgggg tccagcttgc agatcactac cagcagaata ctccgatcgg ggatgggccg
     1201 gttctcctgc ctgacaacca ctacctgtca taccagtcca agctttccaa agatcctaat
     1261 gaaaagcggg atcacatggt cctgctggaa tttgtcaccg ccgccggaat cactctcgga
     1321 atggacgagc tgtacaaagg tgggaccggg ggatctatgg tgagcaaggg cgaagaattg
     1381 ttcactggtg tagtccctat tttggtggaa ctggatgggg atgtcaacgg tcataaattc
     1441 agcgtgcgag gggagggtga gggggacgca actaatggca aattgacact caagctcatc
     1501 tgcacaacgg ggaaactgcc cgtgccatgg cccacactcg ttactactct aggatatggc
     1561 cttatgtgtt tcgcccgcta cccagaccat atgaaacaac acgatttctt taaatcagcc
     1621 atgccagagg ggtacgtcca ggagagaact atcttcttca aggacgatgg taattacaag
     1681 actagggccg aggtgaaatt cgaaggggat acacttgtca accgaatcga gctgaaggga
     1741 atcgacttta aggaggacgg aaacatactc gggcataagt tggaatacaa ctacaacagt
     1801 cacaatgttt acatcacagc cgataagcaa aagaacggca ttaaggctaa ttttaagatc
     1861 cgtcacaaca ttgaggattg agaattcgaa gcttgatccg gctgctaaca aagcccgaaa
     1921 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1981 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat ctggcgtaat
     2041 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2101 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2161 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2221 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2281 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2341 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2401 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2461 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2521 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     2581 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2641 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     2701 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     2761 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     2821 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     2881 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     2941 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     3001 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3061 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     3121 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3181 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     3241 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     3301 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     3361 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     3421 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3481 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3541 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     3601 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3661 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3721 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3781 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3841 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     3901 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3961 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4021 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4081 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4141 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4201 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4261 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4321 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4381 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4441 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     4501 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cag
//
