LOCUS       sequence-314082-        7095 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 7095)
  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 7095)
  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-3 pcDNA3
FEATURES             Location/Qualifiers
     source          1..7095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      686..695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(692..694,695..697,698..1210)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIE"
     CDS             1613..2347
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVRG
                     EGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLGYGLMCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYIMADKQKNGIKANFKIRHNIE"
     promoter        complement(2414..2432)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2415..2432)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2452..2469)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2458..2682
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2728..3156
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2815..2834)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3025..3046
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3165..3185)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3170..3499
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3350..3485
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3412..3431
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3566..4360
                     /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(3620..3639)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4230..4249
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4534..4655
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4571..4590)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4625..4644
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4704..4720)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4704..4720)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4717..4739)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4728..4744
                     /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(4752..4758,4759..4776,4777..4782))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4797..4818
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4935..4952)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5106..5694)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5186..5205)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5865..6656,6657..6725))
                     /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     6488..6507
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6726..6830)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6905..6924)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatcccg cggccgccac catggtgagc aagggcgagg agctgttcac
      721 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
      781 gcgcggcgag ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac
      841 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct ggggcgtgca
      901 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
      961 cgaaggctac gtccaggagc gtaccatctt cttcaaggac gacggcaact acaagacccg
     1021 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1081 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca tcagccacaa
     1141 cgtctatatc accgccgaca agcagaagaa cggcatcaag gcccacttca agatccgcca
     1201 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1261 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca agctgagcaa
     1321 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgcccgcat
     1381 gcaagatgct agcgaagaag agctatcaga gtgctttcgc atttttgaca aggatggaaa
     1441 tggtttcatc gacagggagg aatttggaga catcatccgc cttactggag aacagctcac
     1501 agatgaagat cccgatgaga tatttggaga ttcagacaca gacaaaaatg gaaggattga
     1561 ctttgatgag ttcctgaaga tggtggagaa tgtccagcct ctggccgagc tcatgggcgg
     1621 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1681 gcccgacaac cactacctga gctaccagtc caagctgagc aaagacccca acgagaagcg
     1741 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1801 gctgtacaag ggcggtaccg gcggcagcat ggtgagcaag ggcgaggagc tgttcaccgg
     1861 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgcg
     1921 cggcgagggc gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcaccac
     1981 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgggctacg gcctgatgtg
     2041 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2101 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2161 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2221 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     2281 ctatatcatg gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa
     2341 catcgaggac taagaattct gcagatatcc atcacactgg cggccgctcg agcatgcatc
     2401 tagagggccc tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg
     2461 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2521 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2581 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2641 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     2701 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     2761 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2821 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2881 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2941 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3001 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3061 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3121 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3181 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3241 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3301 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3361 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3421 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     3481 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     3541 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3601 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3661 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3721 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     3781 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     3841 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     3901 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     3961 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4021 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4081 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4141 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4201 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4261 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4321 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4381 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4441 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4501 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4561 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4621 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4681 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4741 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4801 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4861 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4921 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4981 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5041 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5101 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5161 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5221 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5281 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5341 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5401 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5461 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5521 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5581 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5641 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5701 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5761 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5821 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5881 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5941 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6001 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6061 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6121 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6181 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6241 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6301 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6361 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6421 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6481 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6541 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6601 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6661 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6721 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6781 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6841 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     6901 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     6961 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     7021 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     7081 ccagatatac gcgtt
//
