LOCUS       sequence-471239-        5686 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning template for knock-in based strategy with U6 promoter, gRNA 
            and donor tag cloning cassettes, and a fluorescent marker for the 
            plasma membrane under hSyn promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5686)
  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 5686)
  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 KI Cloning Vector_hSyn mTagBFP2-CAAX2
FEATURES             Location/Qualifiers
     source          1..5686
                     /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        2992..3067
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3233..3680
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     intron          3696..3828
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3811..3830
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      3842..3851
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3848..4558
                     /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    4637..5225
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4690..4710)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5108..5119)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5228..5244)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5229..5245)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    5274..5395
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5285..5304)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5339..5358
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5557..5686
                     /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 accgggtctt cgagaagacc tgttttagag
     3001 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     3061 tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta gtccgttttt
     3121 agcgcgtgcg ccaattctgc agacaaatgg ctctagaagc ttgcaagcaa ttgtcgactc
     3181 gagacgcgcc gcttgcggta ccggatccta cgtatctaga gtcgacacta gtagtgcaag
     3241 tgggttttag gaccaggatg aggcggggtg ggggtgccta cctgacgacc gaccccgacc
     3301 cactggacaa gcacccaacc cccattcccc aaattgcgca tcccctatca gagaggggga
     3361 ggggaaacag gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc
     3421 cttcgccccc gcctggcggc gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt
     3481 cactcgccgg tcccccgcaa actccccttc ccggccacct tggtcgcgtc cgcgccgccg
     3541 ccggcccagc cggaccgcac cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc
     3601 tgcgctgcgg cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg
     3661 tgtcgtgcct gagagcgcag tcgaattcaa gctaggtaag tatcaaggtt acaagacagg
     3721 tttaaggaga ccaatagaaa ctgggcttgt cgagacagag atgactcttg cgtttctgat
     3781 aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggt gataagctag
     3841 cgccaccatg gtgtctaagg gcgaagagct gattaaggag aacatgcaca tgaagctgta
     3901 catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc
     3961 ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc
     4021 cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca
     4081 gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac
     4141 cacatacgaa gatgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg
     4201 cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca
     4261 gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct
     4321 ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacgc
     4381 caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta
     4441 tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca
     4501 cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca agctaaatgg
     4561 cggctccgga ggcagcaaga aaaagaagaa gaagtccaag acaaaatgcg tgattatgta
     4621 gtaagagctc atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     4681 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     4741 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     4801 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     4861 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     4921 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     4981 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     5041 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     5101 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     5161 gcctcttccg cgtctacgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     5221 cccgcatcga taccgtcgac ctcgacccgg gcggccgctt cgagcagaca tgataagata
     5281 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     5341 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     5401 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag
     5461 caagtaaaac ctctacaaat gtggtaaaat cgataaggat cttcctagag catggctacg
     5521 tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg atggagttgg
     5581 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     5641 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcag
//
