LOCUS       sequence-254709-        5516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express cre-dependent KASH-EGFP and scramble shRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5516)
  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 5516)
  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-shRNA
FEATURES             Location/Qualifiers
     source          1..5516
                     /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        2676..2751
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   2779..2919
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2779..2908
                     /label=AAV2 ITR
     rep_origin      2994..3449
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3081..3100)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3291..3312
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3466..3485)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3585..3607
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3645..3663)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3731..3835
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3836..3904,3905..4696)
                     /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(4054..4073)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4867..5455
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5356..5375
                     /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 agagcgagct cttctgtttt agtactctgg aaacagaatc
     2701 tactaaaaca aggcaaaatg ccgtgtttat ctcgtcaact tgttggcgag atttttcacg
     2761 tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     2821 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     2881 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     2941 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     3001 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3061 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3121 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3181 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     3241 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3301 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     3361 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3421 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     3481 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3541 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3601 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     3661 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     3721 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3781 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3841 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3901 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3961 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4021 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4081 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4141 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4201 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4261 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4321 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     4381 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4441 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4501 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4561 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4621 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4681 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4741 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4801 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4861 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4921 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4981 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5041 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5101 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5161 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5221 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5281 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5341 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5401 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5461 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
