LOCUS       sequence-471080-        6459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenous tagging of Junctophilin 3 with mEmerald with a 
            fluorescent marker for the plasma membrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6459)
  AUTHORS   Benedetti L, Fan R, Weigel AV, Moore AS, Houlihan PR, Kittisopikul 
            M, Park G, Petruncio A, Hubbard PM, Pang S, Xu CS, Hess HF, Saalfeld
            S, Rangaraju V, Clapham DE, De Camilli P, Ryan TA, 
            Lippincott-Schwartz J
  TITLE     Periodic ER-plasma membrane junctions support long-range Ca(2+) 
            signal integration in dendrites.
  JOURNAL   Cell. 2025 Jan 23;188(2):484-500.e22. doi: 
            10.1016/j.cell.2024.11.029. Epub 2024 Dec 20.
  PUBMED    39708809
REFERENCE   2  (bases 1 to 6459)
  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: 
            "10.1016/j.cell.2024.11.029"; journalName: "Cell"; date: 
            "2025-01-23- 23"; volume: "188"; issue: "2"; pages: "484-500.e22"
COMMENT     Sequence Label: pAAV Jph3gRNA mEmerald hSyn mTagBFP2-CAAX2
FEATURES             Location/Qualifiers
     source          1..6459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(237..825)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(317..336)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(996..1787,1788..1856))
                     /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     1619..1638
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1857..1961)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1987..2442)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2124..2145)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2336..2355
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2514..2643
                     /label=AAV2 ITR
     promoter        2725..2973
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2725..2745
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2896..2915
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3001..3076
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             join(3206..3208,3209..3919)
                     /codon_start=1
                     /product="brightly fluorescent monomeric variant of GFP"
                     /label=mEmerald
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKV
                     NFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3248..3269)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3509..3528)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3856..3877
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        4006..4453
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     intron          4469..4601
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4584..4603
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      4615..4624
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4621..5331
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    5410..5998
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5463..5483)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5881..5892)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6001..6017)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6002..6018)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    6047..6168
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6058..6077)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6112..6131
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6330..6459
                     /label=AAV2 ITR
ORIGIN
        1 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
       61 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
      121 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
      181 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
      241 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      301 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      361 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      421 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
      481 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
      541 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
      601 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
      661 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
      721 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
      781 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
      841 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
      901 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
      961 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     1021 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1081 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     1141 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1201 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1261 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1321 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1381 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1441 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1501 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     1561 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     1621 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1681 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1741 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     1801 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     1861 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     1921 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     1981 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2041 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2101 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2161 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2221 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2281 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2341 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2401 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
     2461 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctgcgcg
     2521 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc
     2581 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
     2641 ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc tctaggaaga
     2701 tctctgcaga gggccctgcg tatggagggc ctatttccca tgattccttc atatttgcat
     2761 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
     2821 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     2881 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     2941 gctttatata tcttgtggaa aggacgaaac accggcaccg agccccgagc tgtaccgcaa
     3001 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     3061 ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag
     3121 tccgttttta gcgcgtgcgc caattctgca gacaaatggc tctagaagct tcccttgcgg
     3181 tacagctcgg ggctctggcg gtgtggtgag caagggcgag gagctgttca ccggggtggt
     3241 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     3301 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     3361 gctgcccgtg ccctggccca ccctcgtgac caccttgacc tacggcgtgc agtgcttcgc
     3421 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     3481 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     3541 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     3601 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca aggtctatat
     3661 caccgccgac aagcagaaga acggcatcaa ggtgaacttc aagacccgcc acaacatcga
     3721 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     3781 cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa
     3841 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     3901 catggacgag ctctacaagg gaggtagccc ttgcggtaca gctcggggct ctcgagacgc
     3961 gccgcttgcg gtaccggatc ctacgtatct agagtcgaca ctagtagtgc aagtgggttt
     4021 taggaccagg atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga
     4081 caagcaccca acccccattc cccaaattgc gcatccccta tcagagaggg ggaggggaaa
     4141 caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag tgccttcgcc
     4201 cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga cgtcactcgc
     4261 cggtcccccg caaactcccc ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc
     4321 agccggaccg caccacgcga ggcgcgagat aggggggcac gggcgcgacc atctgcgctg
     4381 cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg
     4441 cctgagagcg cagtcgaatt caagctaggt aagtatcaag gttacaagac aggtttaagg
     4501 agaccaatag aaactgggct tgtcgagaca gagatgactc ttgcgtttct gataggcacc
     4561 tattggtctt actgacatcc actttgcctt tctctccaca ggtgataagc tagcgccacc
     4621 atggtgtcta agggcgaaga gctgattaag gagaacatgc acatgaagct gtacatggag
     4681 ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag
     4741 ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac
     4801 atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc
     4861 cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac
     4921 gaagatgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc
     4981 tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa
     5041 acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc
     5101 agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa cgccaagacc
     5161 acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac
     5221 tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg
     5281 gcagtggcca gatactgcga cctccctagc aaactggggc acaagctaaa tggcggctcc
     5341 ggaggcagca agaaaaagaa gaagaagtcc aagacaaaat gcgtgattat gtagtaagag
     5401 ctcatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     5461 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     5521 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     5581 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     5641 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     5701 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     5761 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     5821 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     5881 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     5941 ccgcgtctac gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     6001 cgataccgtc gacctcgacc cgggcggccg cttcgagcag acatgataag atacattgat
     6061 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     6121 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     6181 tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagtaa
     6241 aacctctaca aatgtggtaa aatcgataag gatcttccta gagcatggct acgtagataa
     6301 gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc
     6361 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     6421 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcag
//
