LOCUS       sequence-339899-        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-2B pcDNA3
FEATURES             Location/Qualifiers
     source          1..7104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     241..261
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(270..698)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(380..401)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     592..611
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    744..968
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     957..974
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        994..1012
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     994..1011
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2418..2437
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2677..2698
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2740..2749
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2787..2806)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2788..2806)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2851..3054)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2880..2900)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3055..3434
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3606..3625
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3700..3804
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3805..3873,3874..4665)
                     /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(4023..4042)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4836..5424
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5325..5344
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5578..5595
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5712..5733
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5748..5753,5754..5771,5772..5778)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5786..5802
                     /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)."
     primer_bind     5791..5813
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5810..5826
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5810..5826
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5875..5996
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5886..5905)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5940..5959
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(6170..6964)
                     /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(6281..6300)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6891..6910
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(7031..7104,1..256))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(7045..7104,1..76)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(join(7099..7104,1..14))
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg
       61 gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat
      121 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg
      181 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
      241 accctaactg acacacattc cacagaatta attcgcgtta aatttttgtt aaatcagctc
      301 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      361 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      421 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      481 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      541 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      601 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      661 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg ggataccccc tagagcccca
      721 gctggttctt tccgcctcag aagccataga gcccaccgca tccccagcat gcctgctatt
      781 gtcttcccaa tcctccccct tgctgtcctg ccccacccca ccccccagaa tagaatgaca
      841 cctactcaga caatgcgatg caatttcctc attttattag gaaaggacag tgggagtggc
      901 accttccagg gtcaaggaag gcacggggga ggggcaaaca acagatggct ggcaactaga
      961 aggcacagtc gaggctgatc agcgagctct agcatttagg tgacactata gaatagggcc
     1021 ctctagatgc atgctcgagc ggccgccagt gtgatggata tctgcagaat tctcaatcct
     1081 caatgttgtg acggatctta aaattagcct taatgccgtt cttttgctta tcggctgtga
     1141 tgtaaacatt gtgactgttg tagttgtatt ccaacttatg cccgagtatg tttccgtcct
     1201 ccttaaagtc gattcccttc agctcgattc ggttgacaag tgtatcccct tcgaatttca
     1261 cctcggccct agtcttgtaa ttaccatcgt ccttgaagaa gatagttctc tcctggacgt
     1321 acccctctgg catggctgat ttaaagaaat cgtgttgttt catatggtct gggtagcggg
     1381 cgaaacacat aaggccatat cctagagtag taacgagtgt gggccatggc acgggcagtt
     1441 tccccgttgt gcagatgagc ttgagtgtca atttgccatt agttgcgtcc ccctcaccct
     1501 cccctcgcac gctgaattta tgaccgttga catccccatc cagttccacc aaaataggga
     1561 ctacaccagt gaacaattct tcgcccttgc tcaccataga tcccccggtc ccacctttgt
     1621 acagctcgtc cattccgaga gtgattccgg cggcggtgac aaattccagc aggaccatgt
     1681 gatcccgctt ttcattagga tctttggaaa gcttggactg gtatgacagg tagtggttgt
     1741 caggcaggag aaccggccca tccccgatcg gagtattctg ctggtagtga tctgcaagct
     1801 ggaccccacc catgagctcg gggtagatgg gctgcacgtt ttccaccatc ttcaggaact
     1861 cgtcgaaatc gattctgccg ttcttgtcgg tgtcggagtc gccgaagatc tcgtccacgt
     1921 cctcgtcggt cagctgctcg ccggtcagtc tgatgatgtc gccaaactcc tctctgtcga
     1981 tgaagccgtt gccgtcgaag tcgaagattc tgaagcactc gctcagttcc tcttcgctgg
     2041 cgtcggcaac ttgcatgcgg gcggcggtca cgaactccag caggaccatg tgatcgcgct
     2101 tctcgttggg gtctttgctc agcttggact gggtgctcag gtagtggttg tcgggcagca
     2161 gcacggggcc gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc
     2221 cgtcctcgat gttgcagttg aggccgaagt tggccttgat gccgttcttc tgcttgtcgg
     2281 cggtgatata gacgttgccg tggatggcgt tgtactccag cttgtgcccc aggatgttgc
     2341 cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga
     2401 acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct
     2461 ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt
     2521 agcgggcgaa gcactgcacg ccccagctca gggtggtcac gagggtgggc cagggcacgg
     2581 gcagcttgcc ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct
     2641 cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga
     2701 tgggcaccac cccggtgaac agctcctcgc ccttgctcac catggtggcg gccgcgggat
     2761 ccgagctcgg taccaagctt gggtctccct atagtgagtc gtattaattt cgataagcca
     2821 gtaagcagtg ggttctctag ttagccagag agctctgctt atatagacct cccaccgtac
     2881 acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg
     2941 ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc
     3001 ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt
     3061 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
     3121 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
     3181 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
     3241 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
     3301 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
     3361 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt
     3421 caataatcaa tgtcaacgcg tatatctggc ccgtacatcg cgaagcagcg caaaacgcct
     3481 aaccctaagc agattcttca tgcaattgtc ggtcaagcct tgccttgttg tagcttaaat
     3541 tttgctcgcg cactactcag cgacctccaa cacacaagca gggagcagat actggcttaa
     3601 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aggggatcgg gagatctccc
     3661 gatccgtcga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3721 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3781 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     3841 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     3901 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     3961 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4021 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4081 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4141 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4201 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4261 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4321 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     4381 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4441 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4501 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     4561 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4621 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     4681 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4741 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4801 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4861 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4921 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4981 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5041 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5101 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5161 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5221 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5281 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5341 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5401 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5461 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5521 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     5581 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     5641 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     5701 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     5761 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     5821 tatgaccatg attacgccaa gctctagcta gaggtcgacg gtatacagac atgataagat
     5881 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     5941 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttgggg
     6001 tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg
     6061 aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat
     6121 ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact
     6181 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     6241 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     6301 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     6361 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct
     6421 cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     6481 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
     6541 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     6601 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     6661 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     6721 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
     6781 cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
     6841 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
     6901 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
     6961 tcatgcgaaa cgatcctcat cctgtctctt gatcagatcc gaaaatggat atacaagctc
     7021 ccgggagctt tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata
     7081 gctcagaggc agaggcggcc tcgg
//
