LOCUS       sequence-214987-        3825 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 driven SaCas9 gRNA expression cassette without a gRNA. SapI can 
            be used to clone in gRNAs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3825)
  AUTHORS   Kumar N, Stanford W, de Solis C, Aradhana, Abraham ND, Dao TJ, 
            Thaseen S, Sairavi A, Gonzalez CU, Ploski JE
  TITLE     The Development of an AAV-Based CRISPR SaCas9 Genome Editing System 
            That Can Be Delivered to Neurons in vivo and Regulated via 
            Doxycycline and Cre-Recombinase.
  JOURNAL   Front Mol Neurosci. 2018 Nov 13;11:413. doi: 
            10.3389/fnmol.2018.00413. eCollection 2018.
  PUBMED    30483052
REFERENCE   2  (bases 1 to 3825)
  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: "Front Mol 
            Neurosci."; date: "2018-11-13"; volume: "11:413. doi"; pages: " 
            10.3389/fnmol.2018.00413. eCollection 2018"
COMMENT     Sequence Label: pJEP316-pAAV-U6SaCas9gRNA(SapI)-pA  Empty Cass...
FEATURES             Location/Qualifiers
     source          1..3825
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        156..404
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     156..176
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     327..346
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        423..498
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     polyA_signal    572..1048
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(771..790)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   1088..1228
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1088..1217
                     /label=AAV2 ITR
     rep_origin      1303..1758
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1390..1409)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1600..1621
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1775..1794)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1894..1916
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1954..1972)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2040..2144
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2145..2213,2214..3005)
                     /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(2363..2382)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3176..3764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3665..3684
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt
      181 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      361 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggaaga gcgagctctt
      421 ctgttttagt actctggaaa cagaatctac taaaacaagg caaaatgccg tgtttatctc
      481 gtcaacttgt tggcgagatt tttaccggtc catggactag ttctagtcga tatcaagctt
      541 atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc cctccccagt
      601 gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt
      661 aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt
      721 ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga
      781 accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca
      841 agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct
      901 cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc
      961 caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc
     1021 gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt gcggaccgag
     1081 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     1141 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     1201 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     1261 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     1321 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1381 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1441 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1501 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1561 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1621 gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     1681 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1741 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     1801 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     1861 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     1921 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     1981 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2041 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2101 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2161 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2221 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2281 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2341 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     2401 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2461 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2521 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2581 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2641 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     2701 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2761 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2821 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     2881 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     2941 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3001 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3061 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3121 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3181 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3241 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3301 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3361 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3421 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3481 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3541 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3601 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3661 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3721 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3781 cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
