LOCUS       sequence-426324-        7278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Neuron specific expression of a Cre-dependent EYFP tagged to the 
            nuclear membrane through the KASH domain, with an empty gRNA 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7278)
  AUTHORS   Apelblat D, Roethler O, Bitan L, Keren-Shaul H, Spiegel I
  TITLE     Meso-seq for in-depth transcriptomics in ultra-low amounts of 
            FACS-purified neuronal nuclei.
  JOURNAL   Cell Rep Methods. 2022 Jul 25;2(8):100259. doi: 
            10.1016/j.crmeth.2022.100259. eCollection 2022 Aug 22.
  PUBMED    36046622
REFERENCE   2  (bases 1 to 7278)
  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"; journalName: "Cell Rep 
            Methods."; date: "2022-07-25"; volume: "2(8):100259. doi"; pages: " 
            10.1016/j.crmeth.2022.100259. eCollection 2022 Aug 22"
COMMENT     Sequence Label: AAV-U6-sgRNA-hSyn1-DIO-EYFP-KASH
FEATURES             Location/Qualifiers
     source          1..7278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        157..405
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     157..177
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     328..347
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        424..499
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        541..988
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(1019..1052)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1103..1136)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1412..2122,2123..2125,2126..2128))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      2125..2134
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2141..2174
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2225..2258
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(2534..3244,3245..3247,3248..3250))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      3247..3256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3263..3296
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3347..3380
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3405..3993
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3458..3478)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3876..3887)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4025..4501
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4224..4243)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4541..4681
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4541..4670
                     /label=AAV2 ITR
     rep_origin      4756..5211
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4843..4862)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5053..5074
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5228..5247)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5347..5369
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5407..5425)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5493..5597
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5598..5666,5667..6458)
                     /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     complement(5816..5835)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6629..7217
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7118..7137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagagg gcctatttcc catgattcct
      181 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
      241 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
      301 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
      361 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaag agcgagctct
      421 tctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
      481 agtggcaccg agtcggtgct ttttttctag actgcagagg gccctgcgta tgtgcgtatg
      541 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga
      601 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga
      661 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg
      721 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc
      781 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg
      841 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcac gagatagggg ggcacgggcg
      901 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg
      961 aggagtcgtg tcgtgcctga gagcgcaggg taccggatcc tctagagtcg actccggaat
     1021 aacttcgtat aggatacttt atacgaagtt atgcagaatg gtagctggat tgtagctgct
     1081 attagcaata tgaaacctct taataacttc gtatagcata cattatacga agttatggcg
     1141 cgccttaggt gggaggtggc ccgttggtgt accgcagcat ggggtagaag gatcgggcaa
     1201 agttgttggc ctgggtgcag ctgtagtcat cttcagaggc aggtagcagg caggccagga
     1261 gcagcagcag cagcagaagc agctgcaacg gtagcgctgc cctgatcacc cttgagagga
     1321 aggagcgtct cggctgggag ctgccggggc tgtcgaggtg gggcatcctg ctgtctgtct
     1381 cctcctcctc ctctcgagat ctgagtccgg acttgtacag ctcgtccatg ccgagagtga
     1441 tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt
     1501 tgctcagggc ggactggtag ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc
     1561 cgatgggggt gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt
     1621 ggcggatctt gaagttcacc ttgatgccgt tcttctgctt gtcggccatg atatagacgt
     1681 tgtggctgtt gtagttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt
     1741 cgatgccctt cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc
     1801 gggtcttgta gttgccgtcg tccttgaaga agatggtgcg ctcctggacg tagccttcgg
     1861 gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg gcgaagcact
     1921 gcaggccgta gccgaaggtg gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg
     1981 tgcagatgaa cttcagggtc agcttgccgt aggtggcatc gccctcgccc tcgccggaca
     2041 cgctgaactt gtggccgttt acgtcgccgt ccagctcgac caggatgggc accaccccgg
     2101 tgaacagctc ctcgcccttg ctcaccatgg tggcgctagc ataacttcgt ataaagtatc
     2161 ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta gaatggtgca
     2221 aagaataact tcgtataatg tatgctatac gaagttatgg cgcgccttag gtgggaggtg
     2281 gcccgttggt gtaccgcagc atggggtaga aggatcgggc aaagttgttg gcctgggtgc
     2341 agctgtagtc atcttcagag gcaggtagca ggcaggccag gagcagcagc agcagcagaa
     2401 gcagctgcaa cggtagcgct gccctgatca cccttgagag gaaggagcgt ctcggctggg
     2461 agctgccggg gctgtcgagg tggggcatcc tgctgtctgt ctcctcctcc tcctctcgag
     2521 atctgagtcc ggacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga
     2581 actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggt
     2641 agctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct
     2701 ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca
     2761 ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt
     2821 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
     2881 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
     2941 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
     3001 agtcgtgctg cttcatgtgg tcggggtagc gggcgaagca ctgcaggccg tagccgaagg
     3061 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
     3121 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
     3181 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
     3241 tgctcaccat ggtggcgcta gcataacttc gtataaagta tcctatacga agttatttgc
     3301 cttaacccag aaattatcac tgttattctt tagaatggtg caaagaataa cttcgtataa
     3361 tgtatgctat acgaagttat gaattcgata tcaagcttat cgataatcaa cctctggatt
     3421 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3481 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3541 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     3601 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     3661 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     3721 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3781 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct
     3841 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3901 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     3961 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgagcgctg ctcgagagat
     4021 ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac
     4081 tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt
     4141 gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa
     4201 gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc
     4261 ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga
     4321 gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag
     4381 acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc
     4441 accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct
     4501 tctgattttg taggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag
     4561 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     4621 cgacgcccgg gctttgtccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     4681 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     4741 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4801 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     4861 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4921 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     4981 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5041 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     5101 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     5161 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     5221 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     5281 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5341 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     5401 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     5461 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5521 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5581 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5641 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5701 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5761 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5821 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5881 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5941 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     6001 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6061 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6121 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6181 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6241 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6301 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6361 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6421 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6481 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6541 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6601 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6661 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6721 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6781 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6841 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6901 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6961 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     7021 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7081 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7141 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7201 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7261 ggccttttgc tcacatgt
//
