LOCUS       sequence-254733-        5553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express cre-dependent KASH-EGFP and Gmeb1 shRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5553)
  AUTHORS   Tuesta LM, Djekidel MN, Chen R, Lu F, Wang W, Sabatini BL, Zhang Y
  TITLE     In vivo nuclear capture and molecular profiling identifies Gmeb1 as 
            a transcriptional regulator essential for dopamine neuron function.
  JOURNAL   Nat Commun. 2019 Jun 7;10(1):2508. doi: 10.1038/s41467-019-10267-0.
  PUBMED    31175277
REFERENCE   2  (bases 1 to 5553)
  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: "Nat 
            Commun."; date: "2019-06-7"; volume: "10(1):2508. doi"; pages: " 
            10.1038/s41467-019-10267-0"
COMMENT     Sequence Label: AAV-phsyn-FLEX-KASH-EGFP-U6-ShGmeb1
FEATURES             Location/Qualifiers
     source          1..5553
                     /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        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(660..693)
                     /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(744..777)
                     /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(1053..1763,1764..1766,1767..1769))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1053..1763,1764..1766,1767..1769))
                     /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(1095..1116)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1444..1463
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1703..1724
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    1776..1809
                     /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    1860..1893
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1920..2396
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2119..2138)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     promoter        2409..2657
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2409..2429
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2580..2599
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2713..2788
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   2816..2956
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2816..2945
                     /label=AAV2 ITR
     rep_origin      3031..3486
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3118..3137)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3328..3349
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3503..3522)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3622..3644
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3682..3700)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3768..3872
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3873..3941,3942..4733)
                     /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(4091..4110)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4904..5492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5393..5412
                     /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 gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgccctagg tgggaggtgg cccgttggtg taccgcagca tggggtagaa ggatcgggca
      841 aagttgttgg cctgggtgca gctgtagtca tcttcagagg caggtagcag gcaggccagg
      901 agcagcagca gcagcagaag cagctgcaac ggtagcgctg ccctgatcac ccttgagagg
      961 aaggagcgtc tcggctggga gctgccgggg ctgtcgaggt ggggcatcct gctgtctgtc
     1021 tcctcctcct cctctcgaga tctgagtccg gacttgtaca gctcgtccat gccgagagtg
     1081 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
     1141 ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
     1201 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg
     1261 tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg
     1321 ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag
     1381 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg
     1441 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg
     1501 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac
     1561 tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg
     1621 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac
     1681 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg
     1741 gtgaacagct cctcgccctt gctcaccatg ctagcataac ttcgtataaa gtatcctata
     1801 cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagaa
     1861 taacttcgta taatgtatgc tatacgaagt tatgaattcg atctgctcga gagatctacg
     1921 ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag
     1981 tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct
     2041 tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa
     2101 cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc
     2161 tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt
     2221 tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg
     2281 gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt
     2341 ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttctct gtccttctga
     2401 ttttgtagga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     2461 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     2521 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     2581 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     2641 ggaaaggacg aaacaccgga ttactcctgt gggccagtcc ctcgagggac tggcccacag
     2701 gagtaatttt ttgttttagt actctggaaa cagaatctac taaaacaagg caaaatgccg
     2761 tgtttatctc gtcaacttgt tggcgagatt tttcacgtgc ggaccgagcg gccgcaggaa
     2821 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     2881 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     2941 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3001 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     3061 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     3121 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3181 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3241 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3301 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3361 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     3421 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     3481 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     3541 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3601 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3661 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     3721 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3781 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3841 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     3901 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     3961 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     4021 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     4081 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     4141 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     4201 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     4261 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     4321 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     4381 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     4441 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     4501 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     4561 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     4621 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     4681 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     4741 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     4801 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     4861 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4921 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4981 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5041 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5101 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5161 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5221 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5281 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5341 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5401 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5461 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5521 tcctggcctt ttgctggcct tttgctcaca tgt
//
