LOCUS       sequence-237603-        6971 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector that expresses guide RNAs targeting rat TH and 
            expresses EGFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6971)
  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 6971)
  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: pOTTC1088 - pAAV TH gRNA A+B pair EF1a EGFP
FEATURES             Location/Qualifiers
     source          1..6971
                     /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        722..1900
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          953..1891
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1848..1868
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1920..1929
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1926..1928,1929..1931,1932..2645)
                     /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(1926..1928,1929..1931,1932..2642)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1971..1992)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2232..2251)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2579..2600
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2679..3267
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2732..2752)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3150..3161)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3299..3775
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3498..3517)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     promoter        3858..4106
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3858..3878
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4029..4048
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4128..4203
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   4234..4374
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4234..4363
                     /label=AAV2 ITR
     rep_origin      4449..4904
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4536..4555)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4746..4767
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4921..4940)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5040..5062
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5100..5118)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5186..5290
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5291..5359,5360..6151)
                     /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(5509..5528)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6322..6910
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6811..6830
                     /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 tctaaaacaa gggcttcaga agggccgtca 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 ctatagtgag cgcgtaagct ttgcaaagat ggataaagtt
      661 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      721 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      781 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      841 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      901 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      961 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
     1021 tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg
     1081 ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg
     1141 gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc
     1201 tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg
     1261 gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc
     1321 cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg
     1381 agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc
     1441 tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc
     1501 agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag
     1561 gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
     1621 gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga
     1681 ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat
     1741 ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta
     1801 attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac
     1861 agtggttcaa agtttttttc ttccatttca ggtgtcgtga ggtaccggat ccaccggtcg
     1921 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1981 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     2041 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2101 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     2161 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2221 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2281 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2341 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     2401 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2461 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2521 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2581 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2641 agtaaagcgg ccgcactcga gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     2701 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     2761 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     2821 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     2881 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     2941 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3001 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3061 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc
     3121 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     3181 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     3241 ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg
     3301 gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt
     3361 gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt
     3421 ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac
     3481 ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct
     3541 cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt
     3601 gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg
     3661 tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg
     3721 gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat
     3781 tttgtaggta accacctcac tatagggcga attgggtacc cgtctcacag gcggatcgat
     3841 ccaaggtcgg gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat
     3901 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     3961 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     4021 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     4081 atatcttgtg gaaaggacga aacaccgagg ccgaggctgt cacggtggtt ttagagctag
     4141 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     4201 tgcttttttt ctccgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     4261 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     4321 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     4381 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     4441 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4501 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     4561 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4621 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     4681 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4741 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     4801 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     4861 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     4921 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     4981 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5041 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     5101 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     5161 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5221 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5281 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5341 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5401 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5461 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5521 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5581 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5641 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5701 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5761 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5821 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5881 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5941 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6001 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6061 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6121 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6181 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6241 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6301 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6361 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6421 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6481 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6541 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6601 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6661 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6721 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6781 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6841 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6901 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6961 tgctcacatg t
//
