LOCUS       sequence-237595-        6970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector that expresses guide RNAs targeting rat MANF and 
            expresses EGFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6970)
  AUTHORS   Back S, Necarsulmer J, Whitaker LR, Coke LM, Koivula P, Heathward 
            EJ, Fortuno LV, Zhang Y, Yeh CG, Baldwin HA, Spencer MD, 
            Mejias-Aponte CA, Pickel J, Hoffman AF, Spivak CE, Lupica CR, 
            Underhill SM, Amara SG, Domanskyi A, Anttila JE, Airavaara M, Hope 
            BT, Hamra FK, Richie CT, Harvey BK
  TITLE     Neuron-Specific Genome Modification in the Adult Rat Brain Using 
            CRISPR-Cas9 Transgenic Rats.
  JOURNAL   Neuron. 2019 Feb 8. pii: S0896-6273(19)30062-5. doi: 
            10.1016/j.neuron.2019.01.035.
  PUBMED    30792150
REFERENCE   2  (bases 1 to 6970)
  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: "Neuron. 
            2019 Feb 8. pii: S0896-6273(19)30062-5. doi: 
            10.1016/j.neuron.2019.01.035."
COMMENT     Sequence Label: pOTTC1388 - pAAV MANF gRNA A+B EF1a EGFP
FEATURES             Location/Qualifiers
     source          1..6970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_RNA        complement(183..258)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(281..594)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(342..362)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        721..1899
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          952..1890
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1847..1867
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1919..1928
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1925..1927,1928..1930,1931..2644)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1925..1927,1928..1930,1931..2641)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1970..1991)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2231..2250)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2578..2599
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2678..3266
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2731..2751)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3149..3160)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3298..3774
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3497..3516)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     promoter        3857..4105
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3857..3877
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4028..4047
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4127..4202
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   4233..4373
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4233..4362
                     /label=AAV2 ITR
     rep_origin      4448..4903
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4535..4554)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4745..4766
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4920..4939)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5039..5061
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5099..5117)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5185..5289
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5290..5358,5359..6150)
                     /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(5508..5527)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6321..6909
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6810..6829
                     /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 gcgtaagctg gagctccacc gcggcgtctc agtacaaaaa
      181 aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc
      241 tatttctagc tctaaaacgg cagaacttta taagttctca aacaaggctt ttctccaagg
      301 gatatttata gtctcaaaac acacaattac tttacagtta gggtgagttt ccttttgtgc
      361 tgttttttaa aataataatt tagtatttgt atctcttata gaaatccaag cctatcatgt
      421 aaaatgtagc tagtattaaa aagaacagat tatctgtctt ttatcgcaca ttaagcctct
      481 atagttacta ggaaatatta tatgcaaatt aaccggggca ggggagtagc cgagcttctc
      541 ccacaagtct gtgcgagggg gccggcgcgg gcctagagat ggcggcgtcg gatctatctg
      601 agacgggtac ccaattcgcc ctaagtgagc gcgtaagctt tgcaaagatg gataaagttt
      661 taaacagaga ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt
      721 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
      781 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      841 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      901 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      961 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
     1021 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
     1081 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
     1141 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
     1201 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
     1261 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
     1321 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
     1381 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
     1441 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1501 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1561 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1621 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1681 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1741 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1801 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1861 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc caccggtcgc
     1921 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1981 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     2041 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     2101 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     2161 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     2221 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     2281 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     2341 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     2401 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     2461 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     2521 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     2581 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     2641 gtaaagcggc cgcactcgag atatcaagct tatcgataat caacctctgg attacaaaat
     2701 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     2761 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     2821 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     2881 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     2941 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     3001 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     3061 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct
     3121 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     3181 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     3241 gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga gatctacggg
     3301 tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg
     3361 cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc
     3421 tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc
     3481 tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc
     3541 actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg
     3601 ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt
     3661 ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg
     3721 cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt
     3781 ttgtaggtaa ccacctcact atagggcgaa ttgggtaccc gtctcacagg cggatcgatc
     3841 caaggtcggg caggaagagg gcctatttcc catgattcct tcatatttgc atatacgata
     3901 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     3961 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     4021 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     4081 tatcttgtgg aaaggacgaa acaccgcatc cctgtctttg aggtccgttt tagagctaga
     4141 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     4201 gctttttttc tccgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac
     4261 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4321 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4381 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     4441 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4501 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     4561 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4621 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     4681 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4741 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     4801 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     4861 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     4921 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     4981 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5041 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     5101 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     5161 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5221 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5281 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5341 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5401 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5461 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5521 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5581 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5641 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5701 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5761 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5821 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5881 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5941 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6001 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6061 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6121 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6181 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6241 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6301 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6361 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6421 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6481 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6541 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6601 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6661 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6721 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6781 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6841 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6901 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6961 gctcacatgt
//
