LOCUS       Exported                7308 bp ds-DNA     circular SYN 27-SEP-2021
DEFINITION  Fluorescent reporter for Ca2+ inside primary cilia with mCherry 
            reference.
ACCESSION   .
VERSION     .
KEYWORDS    5HT6-mCherry-G-GECO1.0
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7308)
  AUTHORS   Su S, Phua SC, Derose R, Chiba S, Narita K, Kalugin PN, Katada T, 
            Kontani K, Takeda S, Inoue T
  TITLE     Genetically encoded calcium indicator illuminates calcium dynamics 
            in primary cilia.
  JOURNAL   Nat Methods. 2013 Sep 22. doi: 10.1038/nmeth.2647.
  PUBMED    24056873
REFERENCE   2  (bases 1 to 7308)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 27, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7308
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      537..546
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      1893..1902
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1899..2606
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2044..2062)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2312..2331
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2640..2696
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     2909..2930
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3297..3316)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3432..3875
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYIGAAEL
                     RHVMTNLGEKLTDEEVDEMIRVADIDGDGQVNYEEFVQMMTAK"
     polyA_signal    4036..4157
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4073..4092)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4127..4146
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4164..4619)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4301..4322)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4513..4532
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4646..4750
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4752..5109
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(4775..4795)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4960..5095
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5022..5041
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5144..5938
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5198..5217)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5808..5827
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6126..6145)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6170..6217
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6546..7134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7035..7054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcca
      541 ccatggttcc agagcccggc cctgtcaaca gtagcacccc agcctggggc cccgggccac
      601 cgccagcccc agggggcagc ggctgggtgg ccgctgcgct gtgcgtggtc atcgtgctga
      661 cggcagccgc caattcgctg ctgatcgcgc tcatctgcac gcagcccgcg ctgcgcaaca
      721 cgtctaactt cttcctggtg tcgctcttca cgtcggacct gatggtggga ttggtggtga
      781 tgcccccagc catgctgaac gcgctgtatg ggcgctgggt actagctcgc ggcctctgtc
      841 tgctttggac cgccttcgac gtgatgtgct gcagcgcctc cattctcaac ctctgcctca
      901 tcagcctgga ccgctacctg ctcatcctct cgccgctgcg ctacaagctg cgcatgacag
      961 ccccgcgtgc cctggcgctc atcctgggtg cctggagcct cgccgcgctg gcctccttcc
     1021 tgcccctctt gctgggctgg catgaactgg gcaaagctcg aacatctgcc ccgggccagt
     1081 gccgcctatt ggccagcctg ccttatgtcc tcgtggcgtc cggcgtcacc tttttcctgc
     1141 cttcgggtgc catctgcttc acctactgca ggatcttgct ggctgcccgc aagcaggcgg
     1201 tgcaagtggc ctcgctcacc acgggcacgg ctacggctgg ccaggccttg gaaaccttgc
     1261 aggtgcccag gacaccacgc ccagggatgg agtctgctga cagtaggcgt ctgaccacca
     1321 agcatagcag gaaggccttg aaggccagcc tgaccctcgg catcctgctg agcatgttct
     1381 ttgtcacctg gctgcccttc tttgtggcca gcatagctca ggccgtatgt gactgcatct
     1441 ctccaggtct cttcgatgtc ctcacatggc tgggatactg taatagcacc atgaacccta
     1501 tcatctatcc cctcttcatg cgggacttca agagggccct gggcaggttc gtgccgtgtg
     1561 tccactgtcc cccggagcac cgggccagcc ccgcctcccc ctccatgtgg acctctcaca
     1621 gtggtgccag gccaggcctc agcctgcagc aggtgctgcc cctgcctctg ccacccaact
     1681 cagattcaga ctcagcttca gggggcacct cgggcctgca gctcacagcc cagcttttgc
     1741 tgcctggaga ggcgacccgg gaccccccgc cacccaccag ggcccctact gtggtcaact
     1801 tcttcgtcac agactctgtg gagcctgaga tacggcagca tccacttggt tcccccatga
     1861 acagtgctgg tggtagcgca ggaggaccgg tcgccaccat ggtaagcaag ggcgaggagg
     1921 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     1981 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     2041 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     2101 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     2161 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     2221 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     2281 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct
     2341 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     2401 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     2461 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     2521 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     2581 ccggcggcat ggacgagctg tacaagtccg gactcagatc caccatggtc gactcatcac
     2641 gtcgtaagtg gaataagaca ggtcacgcag tcagagctat aggtcggctg agctcactcg
     2701 agaacgtcta tatcaaggcc gacaagcaga agaacggcat caaggcgaac ttcaagatcc
     2761 gccacaacat cgaggacggc ggcgtgcagc tcgcctacca ctaccagcag aacaccccca
     2821 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcgtgcag tccatacttt
     2881 cgaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     2941 ggatcactct cggcatggac gagctgtaca agggcggtac cggagggagc atggtgagca
     3001 agggcgagga gctgttcacc ggggtggtgc ccatccaggt cgagctggac ggcgacgtaa
     3061 acggccacaa gttcagcgtg tccggcgagg gtgagggcga tgccacctac ggcaagctga
     3121 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     3181 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     3241 tcttcaagtc cgccatgccc gaaggctaca tccaggagcg caccatcttc ttcaaggacg
     3301 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     3361 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     3421 acaacacgcg tgaccaactg actgaagagc agatcgcaga atttaaagag gctttctccc
     3481 tatttgacaa ggacggggat gggacgataa caaccaagga gctggggacg gtgatgcggt
     3541 ctctggggca gaaccccaca gaagcagagc tgcaggacat gatcaatgaa gtagatgccg
     3601 acggtgacgg cacaatcgac ttccctgagt tcctgacaat gatggcaaga aaaatgaaag
     3661 acacagacag tgaagaagaa attagagaag cgttccgtgt gtttgataag gacggcaatg
     3721 gctacatcgg cgcagcagag cttcgccacg tgatgacaaa ccttggagag aagttaacag
     3781 atgaagaggt tgatgaaatg atcagggtag cagacatcga tggggatggt caggtaaact
     3841 acgaagagtt tgtacaaatg atgacagcga agtaagaatt ctgcagtcga cggtaccgcg
     3901 ggcccgggat ccaccggatc tagataactg atcataatca gccataccac atttgtagag
     3961 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     4021 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4081 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4141 ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     4201 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     4261 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     4321 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     4381 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     4441 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     4501 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     4561 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     4621 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4681 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4741 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     4801 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4861 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4921 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4981 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     5041 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     5101 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     5161 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     5221 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     5281 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     5341 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     5401 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     5461 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     5521 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     5581 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     5641 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     5701 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     5761 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     5821 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     5881 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     5941 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     6001 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     6061 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     6121 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     6181 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     6241 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     6301 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     6361 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     6421 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6481 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6541 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6601 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6661 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6721 cacttcaaga actctgtatc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6781 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6841 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6901 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6961 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7021 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7081 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7141 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7201 cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta gttattaata
     7261 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccg
//
