LOCUS       sequence-214993-        6300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dox-inducible U6 driven SaCas9 gRNA expression cassette with a gRNA 
            targeting EMX1. Followed by an EFS driven GFP-KASH in a separate 
            reading frame..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6300)
  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 6300)
  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: 
            pJEP322-pAAV-FullU6TO-SaCa9sgRNAi(emx1-sg1)-CMV-TetR-2A-EGFP-KASH-WP
            RE-shortPA
FEATURES             Location/Qualifiers
     source          1..6300
                     /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
     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"
     protein_bind    357..375
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    385..403
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        427..502
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        535..838
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        839..1042
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     993..1013
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1039..1063
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1187..1810
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(1263..1282)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1829..1882
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1883..1885,1886..2596)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1883..1885,1886..2596)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1925..1946)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2186..2205)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2533..2554
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2888..3476
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2941..2961)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3359..3370)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3563..3703
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3563..3692
                     /label=AAV2 ITR
     rep_origin      3778..4233
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3865..3884)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4075..4096
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4250..4269)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4369..4391
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4429..4447)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4515..4619
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4620..4688,4689..5480)
                     /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(4838..4857)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5651..6239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6140..6159
                     /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 tgaaactccc
      361 tatcagtgat agagattata tatctcccta tcagtgatag agaccggcct ccccaaagcc
      421 tggccagttt tagtactctg gaaacagaat ctactaaaac aaggcaaaat gccgtgttta
      481 tctcgtcaac ttgttggcga gatttttacc ggtccatgga ctagttctag agtccgttac
      541 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      601 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      661 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      721 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      781 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      841 gatgcggttt tggcagtaca tcaatgggcg tgaatagcgg tttgactcac ggggatttcc
      901 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      961 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     1021 ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc
     1081 acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgcg gccgggaacg
     1141 gtgcattgga acgctgcagg aattgatccg cggccgcacc ggtaccatga tgtccagatt
     1201 agataaaagt aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg
     1261 tttaacaacc cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca
     1321 tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt gagatgttag ataggcacca
     1381 tactcacttt tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa
     1441 aagttttaga tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg
     1501 gcctacagaa aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg
     1561 tttttcacta gagaatgcat tatatgcact cagcgctgtg gggcatttta ctttaggttg
     1621 cgtattggaa gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga
     1681 tagtatgccg ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc
     1741 agccttctta ttcggccttg aattgatcat atgcggatta gaaaaacaac ttaaatgtga
     1801 aagtgggtcc gcggctagcg gcagtggaga gggcagagga agtctgctaa catgcggtga
     1861 cgtcgaggag aatcctggcc cagtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1921 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1981 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2041 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2101 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2161 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2221 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2281 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2341 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2401 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2461 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     2521 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     2581 ggacgagctg tacaagtccg gactcagatc tcgagaggag gaggaggaga cagacagcag
     2641 gatgccccac ctcgacagcc ccggcagctc ccagccgaga cgctccttcc tctcaagggt
     2701 gatcagggca gcgctaccgt tgcagctgct tctgctgctg ctgctgctcc tggcctgcct
     2761 gctacctgcc tctgaagatg actacagctg cacccaggcc aacaactttg cccgatcctt
     2821 ctaccccatg ctgcggtaca ccaacgggcc acctcccacc taggaattcg atatcaagct
     2881 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     2941 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3001 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3061 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3121 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3181 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3241 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     3301 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3361 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3421 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     3481 ataccgagcg ctaataaaag atctttattt tcattagatc tgtgtgttgg ttttttgtgt
     3541 cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     3601 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     3661 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     3721 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     3781 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3841 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3901 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3961 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     4021 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4081 tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag
     4141 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4201 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     4261 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4321 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4381 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     4441 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4501 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4561 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4621 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4681 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4741 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4801 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4861 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4921 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4981 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5041 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5101 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5161 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5221 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5281 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5341 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5401 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5461 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5521 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5581 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5641 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5701 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5761 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     5821 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5881 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5941 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6001 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6061 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6121 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6181 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6241 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
