LOCUS       Exported                6292 bp ds-DNA     circular SYN 13-MAY-2021
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    
                pJEP322-pAAV-FullU6TO-SaCa9sgRNAi(emx1-sg1)-CMV-TetR-2A-E...
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6292)
  AUTHORS   Kumar N, Stanford W, Aradhana CS, 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) 13
REFERENCE   2  (bases 1 to 6292)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6292
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     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"
     protein_bind    346..364
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    374..392
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        416..491
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        524..827
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        828..1031
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     982..1002
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1028..1052
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1182..1799
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /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="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     primer_bind     complement(1255..1274)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1821..1874
                     /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             1875..2588
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1917..1938)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2178..2197)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2525..2546
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2880..3468
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2933..2953)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3351..3362)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3555..3695
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3555..3684
                     /label=AAV2 ITR
     rep_origin      3770..4225
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3857..3876)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4067..4088
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4242..4261)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4361..4383
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4421..4439)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4507..4611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4612..5472
                     /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(4830..4849)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5643..6231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6132..6151
                     /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 gaaactccct atcagtgata
      361 gagattatat atctccctat cagtgataga gaccggcctc cccaaagcct ggccagtttt
      421 agtactctgg aaacagaatc tactaaaaca aggcaaaatg ccgtgtttat ctcgtcaact
      481 tgttggcgag atttttaccg gtccatggac tagttctaga gtccgttaca taacttacgg
      541 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      601 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      661 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      721 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      781 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      841 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      901 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      961 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1021 taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg
     1081 acctccatag aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg tgcattggaa
     1141 cggacctgca ggaattgatc cgcggccgca ccggtaccat gatgtccaga ttagataaaa
     1201 gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa
     1261 cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa
     1321 ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact
     1381 tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta
     1441 gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag
     1501 aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac
     1561 tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg
     1621 aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc
     1681 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct
     1741 tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt
     1801 ccgcggctag cggcagtgga gagggcagag gaagtctgct aacatgcggt gacgtcgagg
     1861 agaatcctgg cccagtgagc aagggcgagg agctgttcac cggggtggta cccatcctgg
     1921 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1981 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     2041 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     2101 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     2161 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     2221 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     2281 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     2341 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     2401 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2461 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     2521 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2581 tgtacaagtc cggactcaga tctcgagagg aggaggagga gacagacagc aggatgcccc
     2641 acctcgacag ccccggcagc tcccagccga gacgctcctt cctctcaagg gtgatcaggg
     2701 cagcgctacc gttgcagctg cttctgctgc tgctgctgct cctggcctgc ctgctacctg
     2761 cctctgaaga tgactacagc tgcacccagg ccaacaactt tgcccgatcc ttctacccca
     2821 tgctgcggta caccaacggg ccacctccca cctaggaatt cgatatcaag cttatcgata
     2881 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     2941 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     3001 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     3061 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     3121 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     3181 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     3241 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     3301 cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     3361 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     3421 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag
     3481 cgctaataaa agatctttat tttcattaga tctgtgtgtt ggttttttgt gtcacgtgcg
     3541 gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     3601 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     3661 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     3721 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     3781 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3841 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3901 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3961 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4021 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4081 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     4141 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4201 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     4261 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4321 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4381 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4441 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4501 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4561 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4621 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4681 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4741 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4801 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4861 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4921 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4981 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5041 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5101 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5161 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5221 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5281 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5341 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5401 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5461 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5521 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5581 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5641 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5701 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5761 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5821 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5881 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5941 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6001 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6061 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
