LOCUS       sequence-225305-        6907 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express an optogenetic tether to label and interrogate ER-plasma 
            membrane contact sites (MCS); visualized by GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6907)
  AUTHORS   Jing J, He L, Sun A, Quintana A, Ding Y, Ma G, Tan P, Liang X, Zheng
            X, Chen L, Shi X, Zhang SL, Zhong L, Huang Y, Dong MQ, Walker CL, 
            Hogan PG, Wang Y, Zhou Y
  TITLE     Proteomic mapping of ER-PM junctions identifies STIMATE as a 
            regulator of Ca(2+) influx.
  JOURNAL   Nat Cell Biol. 2015 Oct;17(10):1339-47. doi: 10.1038/ncb3234. Epub 
            2015 Aug 31.
  PUBMED    26322679
REFERENCE   2  (bases 1 to 6907)
  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: "10.1038/ncb3234"; 
            journalName: "Nat Cell Biol"; date: "2015-10"; volume: "17"; issue: 
            "10"; pages: "1339-47"
COMMENT     Sequence Label: GFP-LiMETER-v1
FEATURES             Location/Qualifiers
     source          1..6907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        91..470
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        471..674
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     625..645
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     719..738
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        719..737
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(877..879,880..882,883..1593)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(877..879,880..882,883..1593)
                     /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(922..943)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1183..1202)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1530..1551
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1792..2220
                     /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(2354..2371)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2360..2584
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2630..3058
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2717..2736)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2927..2948
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3067..3087)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3072..3401
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3252..3387
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3314..3333
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3468..4262
                     /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(3522..3541)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4132..4151
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4436..4557
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4473..4492)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4527..4546
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4606..4622)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4606..4622)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4619..4641)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4630..4646
                     /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(4654..4660,4661..4678,4679..4684))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4699..4720
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4837..4854)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5008..5596)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5088..5107)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5767..6558,6559..6627))
                     /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     6390..6409
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6628..6732)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6807..6826)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
       61 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
      121 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      181 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      241 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      301 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      361 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      421 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      481 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      541 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      601 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      661 atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta
      721 atacgactca ctatagggag acccaagctg gctagcaagc ttatggatgt atgcgtccgt
      781 cttgccctgt ggctcctctg gggactcctc ctgcaccagg gccagagcct cagccatagt
      841 cacagtgaga aggcgacagg aaccagctcg ggtaccatgg tgagcaaggg cgaggagctg
      901 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
      961 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     1021 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     1081 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     1141 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     1201 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     1261 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     1321 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     1381 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     1441 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     1501 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1561 gggatcactc tcggcatgga cgagctgtac aagggcggcc gcagtggtgg gctggataca
     1621 gtgctctttg ggcctcctct cttgactcgc cataatcacc tcaaggactt catgctggtg
     1681 gtgtctatcg ttattggtgt gggcggctgc tggtttgcct atatccagaa ccgttactcc
     1741 aaggagcacg gagcgggcgc tggtgccgga gctggagcag gtgctctcga gttggctact
     1801 acacttgaac gtattgagaa gaactttgtc attactgacc caagattgcc agataatccc
     1861 attatattcg cgtccgatag tttcttgcag ttgacagaat atagccgtga agaaattttg
     1921 ggaagaaact gcaggtttct acaaggtcct gaaactgatc gcgcgacagt gagaaaaatt
     1981 agagatgcca tagataacca aacagaggtc actgttcagc tgattaatta tacaaagagt
     2041 ggtaaaaagt tctggaacct ctttcacttg cagcctatgc gagatcagaa gggagatgtc
     2101 cagtacttta ttggggttca gttggatgga actgagcatg tccgagatgc tgccgagaga
     2161 gagggagtca tgctgattaa gaaaactgca gaaaatattg atgaggcggc aaaagaactt
     2221 agaattctaa tggagaaaaa atctaagccc aaaaacagtg tatggaagag gctaaaatca
     2281 ccattccgga agaagaaaga ttcagtaact tgataaggat cctctagagg gcccgtttaa
     2341 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2401 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2461 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2521 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     2581 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     2641 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2701 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2761 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2821 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2881 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2941 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3001 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     3061 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3121 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3181 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3241 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3301 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     3361 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     3421 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3481 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3541 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3601 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     3661 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3721 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3781 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3841 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3901 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3961 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     4021 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4081 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4141 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4201 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4261 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4321 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4381 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4441 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4501 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4561 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     4621 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4681 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4741 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4801 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4861 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4921 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4981 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5041 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5101 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5161 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5221 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5281 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5341 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5401 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5461 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5521 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5581 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5641 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5701 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5761 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5821 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5881 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5941 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6001 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6061 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6121 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6181 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6241 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6301 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6361 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6421 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6481 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6541 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6601 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6661 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6721 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
     6781 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     6841 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
     6901 acaacaa
//
