LOCUS       sequence-229568-        3814 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 3814)
  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 3814)
  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..3814
                     /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        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        412..487
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     polyA_signal    561..1037
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(760..779)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   1077..1217
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1077..1206
                     /label=AAV2 ITR
     rep_origin      1292..1747
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1379..1398)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1589..1610
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1764..1783)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1883..1905
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1943..1961)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2029..2133
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2134..2202,2203..2994)
                     /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(2352..2371)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3165..3753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3654..3673
                     /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 gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagta
      421 ctctggaaac agaatctact aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt
      481 ggcgagattt ttaccggtcc atggactagt tctagtcgat atcaagctta tcgataccga
      541 gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg
      601 ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca
      661 ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca
      721 aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga
      781 gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
      841 tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt
      901 tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat
      961 ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg
     1021 ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga
     1081 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     1141 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     1201 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     1261 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     1321 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     1381 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1441 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1501 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1561 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1621 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     1681 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     1741 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     1801 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     1861 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     1921 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     1981 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     2041 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2101 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2161 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2221 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2281 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2341 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2401 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2461 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2521 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2581 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2641 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     2701 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2761 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2821 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     2881 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2941 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3001 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3061 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3121 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3181 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3241 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3301 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3361 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3421 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3481 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3541 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3601 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3661 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3721 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3781 ttcctggcct tttgctggcc ttttgctcac atgt
//
