LOCUS       sequence-225307-        6886 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Also termed as OptoPBer; Express an STIM1-derived optical tether to 
            label and interrogate ER-plasma membrane contact sites (MCS) with 
            light; visualized by GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6886)
  AUTHORS   He L, Jing J, Zhu L, Tan P, Ma G, Zhang Q, Nguyen NT, Wang J, Zhou 
            Y, Huang Y
  TITLE     Optical control of membrane tethering and interorganellar 
            communication at nanoscales.
  JOURNAL   Chem Sci. 2017 Aug 1;8(8):5275-5281. doi: 10.1039/c7sc01115f. Epub 
            2017 May 31.
  PUBMED    28959426
REFERENCE   2  (bases 1 to 6886)
  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"; doi: 
            "10.1039/c7sc01115f"; journalName: "Chem Sci"; date: "2017-08-1- 1";
            volume: "8"; issue: "8"; pages: "5275-5281"
COMMENT     Sequence Label: GFP-LiMETER-v2
FEATURES             Location/Qualifiers
     source          1..6886
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        120..499
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        500..703
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     654..674
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     748..767
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        748..766
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(906..908,909..911,912..1622)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(906..908,909..911,912..1622)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(951..972)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1212..1231)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1559..1580
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1821..2249
                     /codon_start=1
                     /gene="NPH1 from Avena sativa"
                     /product="light, oxygen, or voltage domain from phototropin
                     1 of Avena sativa (Herrou and Crosson, 2011)"
                     /label=LOV2
                     /note="photoswitch that undergoes a light-inducible 
                     conformational change"
                     /translation="LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQ
                     YFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAAKEL"
     primer_bind     complement(2362..2379)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2368..2592
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2638..3066
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2725..2744)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2935..2956
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3075..3095)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3080..3409
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3260..3395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3322..3341
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3476..4270
                     /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(3530..3549)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4140..4159
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4444..4565
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4481..4500)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4535..4554
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4614..4630)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4614..4630)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4627..4649)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4638..4654
                     /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(4662..4668,4669..4686,4687..4692))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4707..4728
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4845..4862)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5016..5604)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5096..5115)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5775..6566,6567..6635))
                     /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     6398..6417
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6636..6740)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6815..6834)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
       61 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      121 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      181 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      241 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      301 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      361 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      421 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      481 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      541 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      601 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      661 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
      721 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
      781 ctagcaagct tatggatgta tgcgtccgtc ttgccctgtg gctcctctgg ggactcctcc
      841 tgcaccaggg ccagagcctc agccatagtc acagtgagaa ggcgacagga accagctcgg
      901 gtaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      961 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1021 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1081 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1141 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1201 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1261 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1321 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1381 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1441 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1501 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     1561 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1621 agggcggccg cagtggtggg ctggatacag tgctctttgg gcctcctctc ttgactcgcc
     1681 ataatcacct caaggacttc atgctggtgg tgtctatcgt tattggtgtg ggcggctgct
     1741 ggtttgccta tatccagaac cgttactcca aggagcacgg agcgggcgct ggtgccggag
     1801 ctggagcagg tgctctcgag ttggctacta cacttgaacg tattgagaag aactttgtca
     1861 ttactgaccc aagattgcca gataatccca ttatattcgc gtccgatagt ttcttgcagt
     1921 tgacagaata tagccgtgaa gaaattttgg gaagaaactg caggtttcta caaggtcctg
     1981 aaactgatcg cgcgacagtg agaaaaatta gagatgccat agataaccaa acagaggtca
     2041 ctgttcagct gattaattat acaaagagtg gtaaaaagtt ctggaacctc tttcacttgc
     2101 agcctatgcg agatcagaag ggagatgtcc agtactttat tggggttcag ttggatggaa
     2161 ctgagcatgt ccgagatgct gccgagagag agggagtcat gctgattaag aaaactgcag
     2221 aaaatattga tgaggcggca aaagaactta gaattctaga ctccagccca ggccggaaga
     2281 agtttcccct caaaatcttt aagaagcctc ttaagaagtg ataaggatcc tctagagggc
     2341 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2401 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2461 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     2521 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     2581 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     2641 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2701 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2761 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2821 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2881 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2941 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3001 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3061 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     3121 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     3181 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3241 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3301 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     3361 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     3421 agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat
     3481 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3541 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3601 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     3661 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3721 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3781 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     3841 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     3901 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     3961 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     4021 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4081 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4141 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4201 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4261 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4321 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4381 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4441 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4501 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4561 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     4621 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4681 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4741 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4801 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4861 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4921 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4981 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5041 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5101 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5161 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5221 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5281 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5341 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5401 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5461 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5521 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5581 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5641 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5701 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5761 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5821 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5881 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5941 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6001 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6061 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6121 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6181 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6241 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6301 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6361 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6421 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6481 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6541 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6601 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6661 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6721 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     6781 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6841 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagta
//
