LOCUS       sequence-202519-        7104 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 7104)
  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 7104)
  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-2C pcDNA3
FEATURES             Location/Qualifiers
     source          1..7104
                     /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"
     regulatory      58..67
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(64..66,67..69,70..582)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIE"
     CDS             994..1728
                     /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(1795..1813)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1796..1813)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1833..1850)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1839..2063
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2109..2537
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2196..2215)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2406..2427
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2546..2566)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2551..2880
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2731..2866
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2793..2812
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2947..3741
                     /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(3001..3020)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3611..3630
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3915..4036
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3952..3971)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4006..4025
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4085..4101)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4085..4101)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4098..4120)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4109..4125
                     /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(4133..4139,4140..4157,4158..4163))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4178..4199
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4316..4333)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4487..5075)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4567..4586)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5246..6037,6038..6106))
                     /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     5869..5888
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6107..6211)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6286..6305)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6477..6856
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6857..7060
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7011..7031
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 taatacgact cactataggg agacccaagc ttggtaccga gctcggatcc cgcggccgcc
       61 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
      121 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
      181 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
      241 accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg
      301 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
      361 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
      421 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
      481 cacaagctgg agtacaacta ctttagcgac aacgtctata tcaccgccga caagcagaag
      541 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc
      601 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
      661 cactacctga gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg
      721 gtcctgctgg agttcgtgac cgccgcccgc atgcaagttg ccgacgccag cgaagaggaa
      781 ctgagcgagt gcttcagaat cttcgacttc gacggcaacg gcttcatcga cagagaggag
      841 tttggcgaca tcatcagact gaccggcgag cagctgaccg acgaggacgt ggacgagatc
      901 ttcggcgact ccgacaccga caagaacggc agaatcgatt tcgacgagtt cctgaagatg
      961 gtggaaaacg tgcagcccat ctaccccgag ctcatgggcg gcgtgcagct cgccgaccac
     1021 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1081 agctaccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1141 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggcggtacc
     1201 ggcggcagca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1261 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgc gcggcgaggg cgagggcgat
     1321 gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1381 tggcccaccc tcgtgaccac cctgggctac ggcctgatgt gcttcgcccg ctaccccgac
     1441 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1501 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1561 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1621 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     1681 cagaagaacg gcatcaaggc caacttcaag atccgccaca acatcgagga ctaagaattc
     1741 tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc ctattctata
     1801 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     1861 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     1921 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     1981 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2041 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     2101 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2161 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2221 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2281 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2341 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2401 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2461 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2521 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2581 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     2641 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2701 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     2761 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     2821 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     2881 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     2941 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3001 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3061 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3121 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3181 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3241 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     3301 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     3361 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     3421 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     3481 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     3541 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     3601 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     3661 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     3721 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     3781 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     3841 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     3901 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3961 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4021 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4081 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4141 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4201 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4261 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4321 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4381 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4441 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4501 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4561 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4621 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4681 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4741 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4801 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4861 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4921 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4981 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5041 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5101 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5161 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5221 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5281 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5341 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5401 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5461 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5521 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5581 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5641 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5701 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5761 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5821 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5881 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5941 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6001 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6061 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6121 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6181 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6241 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     6301 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     6361 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     6421 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
     6481 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     6541 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     6601 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     6661 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     6721 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     6781 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     6841 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     6901 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     6961 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     7021 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
     7081 cactgcttac tggcttatcg aaat
//
