LOCUS       sequence-237596-        6816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector that expresses a Cre-dependent guide RNA targeting rat
            TH and expresses EGFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6816)
  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 6816)
  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: pOTTC1179 - pAAV (flox-stop) TH gRNA A EF1a eGFP
FEATURES             Location/Qualifiers
     source          1..6816
                     /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"
     protein_bind    291..324
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     protein_bind    555..588
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     promoter        986..2164
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1217..2155
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2112..2132
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2184..2193
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2190..2192,2193..2195,2196..2909)
                     /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(2190..2192,2193..2195,2196..2906)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2235..2256)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2496..2515)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2843..2864
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2943..3531
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2996..3016)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3414..3425)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3563..4039
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3762..3781)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4079..4219
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4079..4208
                     /label=AAV2 ITR
     rep_origin      4294..4749
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4381..4400)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4591..4612
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4766..4785)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4885..4907
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4945..4963)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5031..5135
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5136..5204,5205..5996)
                     /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(5354..5373)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6167..6755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6656..6675
                     /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 ataacttcgt
      301 ataatttata ctatacgaag ttatggatcc cccgggcgtg gcttggtggt acacctctgt
      361 catcccagta ggtaggtaga ggcagtcact ggagaacctc atcagtgtca ggccaacctg
      421 agctatacag tgagcaacga gctctgctgt caagaaacaa aatagaaaaa acgtgagtga
      481 gcctccaata ggatgttagg gtgcttgtca gttcctctga ggaacgaatt caaaaatcgc
      541 aaaaaacaag gcttataact tcgtataatt tatactatac gaagttatac taactttaca
      601 gttagggtga gttctcgagt gtgctgtttt ttaaaataat aatttagtat ttgtatctct
      661 tatagaaatc caagcctatc atgtaaaatg tagctagtat taaaaagaac agattatctg
      721 tcttttatcg cacattaagc ctctatagtt actaggaaat attatatgca aattaaccgg
      781 ggcaggggag tagccgagct tctcccacaa gtctgtgcga gggggccggc gcgggcctag
      841 agatggcggc gtcggatcta tctgagacgg gtacccaatt cgccctatag tgagcgcgta
      901 agctttgcaa agatggataa agttttaaac agagaggaat ctttgcagct aatggacctt
      961 ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc
     1021 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     1081 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     1141 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     1201 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
     1261 ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc
     1321 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc
     1381 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg
     1441 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg
     1501 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca
     1561 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac
     1621 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
     1681 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc
     1741 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc
     1801 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc
     1861 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta
     1921 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg
     1981 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt
     2041 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc
     2101 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc
     2161 gtgaggtacc ggatccaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg
     2221 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     2281 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     2341 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     2401 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     2461 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     2521 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     2581 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2641 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2701 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2761 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2821 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2881 ctctcggcat ggacgagctg tacaagtaaa gcggccgcac tcgagatatc aagcttatcg
     2941 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3001 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     3061 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     3121 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     3181 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     3241 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3301 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     3361 tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     3421 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     3481 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     3541 gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct
     3601 ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat
     3661 cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag
     3721 caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg
     3781 gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct
     3841 cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt
     3901 tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta
     3961 atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac
     4021 tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag
     4081 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     4141 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     4201 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4261 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     4321 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     4381 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4441 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4501 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     4561 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     4621 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     4681 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     4741 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     4801 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4861 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4921 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     4981 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5041 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5101 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5161 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5221 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5281 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5341 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5401 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5461 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5521 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5581 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     5641 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     5701 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     5761 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     5821 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     5881 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     5941 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6001 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6061 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6121 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6181 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6241 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6301 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6361 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6421 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6481 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6541 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6601 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6661 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6721 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6781 ggttcctggc cttttgctgg ccttttgctc acatgt
//
