LOCUS       Exported                6552 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  An AAV vector that expresses guide RNA targeting rat TH and 
            expresses EGFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    pOTTC1079 - pAAV TH gRNA A EF1a EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6552)
  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 6552)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6552
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1926..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             1926..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"
     repeat_region   3815..3955
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3815..3944
                     /label=AAV2 ITR
     rep_origin      4030..4485
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4117..4136)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4327..4348
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4502..4521)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4621..4643
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4681..4699)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4767..4871
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4872..5732
                     /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(5090..5109)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5903..6491
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6392..6411
                     /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 accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     3841 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     3901 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     3961 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     4021 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4081 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     4141 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4201 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     4261 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4321 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     4381 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     4441 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     4501 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     4561 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4621 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     4681 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     4741 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4801 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4861 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4921 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4981 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5041 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5101 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5161 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5221 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5281 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5341 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5401 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5461 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5521 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5581 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5641 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5701 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5761 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5821 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5881 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5941 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6001 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6061 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6121 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6181 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6241 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6301 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6361 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6421 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6481 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6541 ttgctcacat gt
//
