LOCUS       sequence-497314-        7530 bp    DNA     circular SYN 14-JAN-2026
DEFINITION  An expression construct encoding a CRY2-fused STIM1 fragment 
            (a.a.238-448) linked to EGFP through an IRES2 element..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7530)
  AUTHORS   Kook YH, Lee H, Lee J, Jeong Y, Rho J, Heo WD, Lee S
  TITLE     AAV-compatible optogenetic tools for activating endogenous calcium 
            channels in vivo.
  JOURNAL   Mol Brain. 2023 Oct 17;16(1):73. doi: 10.1186/s13041-023-01061-7.
  PUBMED    37848907
REFERENCE   2  (bases 1 to 7530)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 14, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol 
            Brain."; date: "2023-10-17"; volume: "16(1):73. doi"; pages: " 
            10.1186/s13041-023-01061-7"
COMMENT     Sequence Label: pCMV-EGFP-IRES2-CRY2-STIM1(238-448)
FEATURES             Location/Qualifiers
     source          1..7530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      597..606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(603..605,606..608,609..1322)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(603..605,606..608,609..1319)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(648..669)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(909..928)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1256..1277
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1337..1911,1912..1914,1915..1923)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1504..1521)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1731..1750
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    4318..4439
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4355..4374)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4409..4428
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4446..4901)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4583..4604)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4795..4814
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4928..5032
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5034..5391
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5034..5225
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5057..5077)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5242..5377
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5426..6220
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5426..6220
                     /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(5480..5499)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6090..6109
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6408..6427)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6452..6499
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6828..7416
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7317..7336
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcca
      601 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      661 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
      721 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
      781 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
      841 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
      901 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
      961 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1021 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1081 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1141 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1201 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1261 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1321 aaagcggccg catccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct
     1381 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg
     1441 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt
     1501 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg
     1561 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac
     1621 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg
     1681 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct
     1741 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg
     1801 atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag
     1861 gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca
     1921 accatggtgt acaagccacc ggtcatgaag atggacaaaa agaccatcgt ctggtttcgg
     1981 agagatttga gaatagaaga taatcccgcg ctcgccgccg cggcccacga gggttccgtc
     2041 ttccccgttt tcatttggtg tcctgaagaa gaaggccagt tttatcccgg aagggcctct
     2101 aggtggtgga tgaagcaaag tctggcccat cttagccagt cactgaaagc actgggcagt
     2161 gatcttaccc tgatcaagac acacaatacc atctctgcca tcctcgactg catcagggtg
     2221 accggcgcaa cgaaagtcgt gtttaaccac ctgtacgatc cagttagtct ggtgcgcgac
     2281 cacactgtga aggagaagct ggtggaacgg gggatcagtg tgcagagcta caacggggac
     2341 cttctgtacg agccatggga gatctattgc gagaaaggga aaccgttcac ctccttcaac
     2401 agttactgga agaaatgttt ggatatgtca atagagtccg ttatgttgcc ccctccctgg
     2461 agactgatgc cgattactgc tgctgcagag gccatctggg cctgctccat cgaggaactc
     2521 ggtctggaaa atgaagcaga aaagccaagc aatgcacttc tcactagagc ctggagcccc
     2581 ggctggtcta atgccgacaa gctgcttaac gagttcatcg aaaaacaact gattgactac
     2641 gcgaagaact ccaagaaagt ggtaggtaac tcaactagct tgctctctcc atatctccat
     2701 tttggcgaga tttctgtccg ccatgtattt cagtgcgctc ggatgaaaca gattatctgg
     2761 gctcgcgata aaaacagcga aggcgccgaa agcgccgatc tgttcctgcg agggatcgga
     2821 cttcgggaat actcccggta tatatgtttc aactttccat tcacacacga gcagagtctg
     2881 ttgtcccacc tcaggttctt cccctgggac gccgatgtcg acaaattcaa ggcatggaga
     2941 cagggaagga cagggtaccc actcgtggat gctggcatga gagagctctg ggctacaggc
     3001 tggatgcaca accgcatccg ggtaatcgtg tcctcatttg ctgtcaagtt tctgctcctg
     3061 ccttggaaat ggggaatgaa gtacttttgg gatacccttc tcgacgccga cttggagtgt
     3121 gacattctgg gatggcaata tattagcggg tcaattcctg acggccatga gttggacagg
     3181 ttggacaatc cggccttgca gggagctaag tatgatcccg aaggagagta tattcgacag
     3241 tggctccccg agctggcccg acttcctacg gagtggattc accatccttg ggacgcacca
     3301 ctgacagtgc tcaaggcaag cggggtggag ctgggcacca attacgctaa gcctatagtt
     3361 gatatagata cagcacgcga gctgctggct aaagcgatct ctcgcactcg ggaggcgcag
     3421 attatgatcg gtgctgccgc cagggacccc cccgacctcg acaacgaatt cagtggggga
     3481 ggcggagggg gtggttcagg tggtggtggt ggttcgggtg gtggcggagg gggaggctcc
     3541 ggactcagat ctaaggagca catgaagaag atgatgaagg acttggaggg gttacaccga
     3601 gctgagcaga gtctgcatga ccttcaggaa aggctgcaca aggcccagga ggagcaccgc
     3661 acagtggagg tggagaaggt ccatctggaa aagaagctgc gcgatgagat caaccttgct
     3721 aagcaggaag cccagcggct gaaggagctg cgggagggta ctgagaatga gcggagccgc
     3781 caaaaatatg ctgaggagga gttggagcag gttcgggagg ccttgaggaa agcagagaag
     3841 gagctagaat ctcacagctc atggtatgct ccagaggccc ttcagaagtg gctgcagctg
     3901 acacatgagg tggaggtgca atattacaac atcaagaagc aaaatgctga gaagcagctg
     3961 ctggtggcca aggagggggc tgagaagata aaaaagaaga gaaacacact ctttggcacc
     4021 ttccacgtgg cccacagctc ttccctggat gatgtagatc ataaaattct aacagctaag
     4081 caagcactga gcgaggtgac agcagcattg cgggagcgcc tgcaccgctg gcaacagatc
     4141 gagatcctct gtggcttcca gattgtcaac aaccctggca tccactaggg atccaccgga
     4201 tctagataac tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     4261 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     4321 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4381 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     4441 cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     4501 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     4561 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     4621 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     4681 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     4741 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     4801 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     4861 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     4921 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4981 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     5041 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     5101 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     5161 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     5221 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     5281 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     5341 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     5401 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     5461 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     5521 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     5581 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     5641 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     5701 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     5761 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     5821 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     5881 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     5941 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     6001 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     6061 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     6121 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     6181 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     6241 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     6301 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     6361 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     6421 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     6481 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     6541 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     6601 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     6661 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6721 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6781 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6841 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6901 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6961 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     7021 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7081 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7141 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7201 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7261 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7321 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7381 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7441 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7501 tctgtggata accgtattac cgccatgcat
//
