LOCUS       Exported                5991 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  An AAV genome containing miRNA target sequence (TS) 708-5p to reduce
            expression of the fluorescent protein eYFP in neurons.
ACCESSION   .
VERSION     .
KEYWORDS    CAG-eYFP-3x-miR708-5p-TS
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5991)
  AUTHORS   Challis RC, Ravindra Kumar S, Chan KY, Challis C, Beadle K, Jang MJ,
            Kim HM, Rajendran PS, Tompkins JD, Shivkumar K, Deverman BE, 
            Gradinaru V
  TITLE     Systemic AAV vectors for widespread and targeted gene delivery in 
            rodents.
  JOURNAL   Nat Protoc. 2019 Feb;14(2):379-414. doi: 10.1038/s41596-018-0097-3.
  PUBMED    30626963
REFERENCE   2  (bases 1 to 5991)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5991
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     enhancer        221..524
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        526..803
                     /label=chicken beta-actin promoter
     primer_bind     1133..1155
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1217..1236
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1279..1288
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1285..2004
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1330..1351)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1591..1610)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1938..1959
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2118..2706
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2171..2191)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2589..2600)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2738..3214
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2937..2956)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3254..3394
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3254..3383
                     /label=AAV2 ITR
     rep_origin      3469..3924
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3556..3575)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3766..3787
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3941..3960)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4060..4082
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4120..4138)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4206..4310
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4311..5171
                     /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(4529..4548)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5342..5930
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5831..5850
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaacac tagttattaa tagtaatcaa
      181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      481 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
      541 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
      601 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg
      661 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
      721 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
      781 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
      841 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
      901 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
      961 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagcgccgg caggaaggaa
     1021 atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct
     1081 cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc
     1141 ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt
     1201 ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa
     1261 ttgcggccca acggtaccgc caccatggtg agcaagggcg aggagctgtt caccggggtg
     1321 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1381 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1441 aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcct gcagtgcttc
     1501 gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1561 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1621 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1681 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     1741 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     1801 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1861 cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgccctgag caaagacccc
     1921 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1981 ggcatggacg agctgtacaa gtgagaattc accggtccca gctagattgt aagctcctta
     2041 ctgtacccag ctagattgta agctccttga ctcacccagc tagattgtaa gctccttcgc
     2101 cggcgaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     2161 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     2221 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     2281 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     2341 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     2401 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     2461 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     2521 cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct
     2581 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     2641 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     2701 ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc
     2761 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat
     2821 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag
     2881 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta
     2941 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg
     3001 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac
     3061 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct
     3121 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt
     3181 acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg
     3241 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     3301 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     3361 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     3421 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     3481 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3541 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3601 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3661 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     3721 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3781 aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc
     3841 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3901 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     3961 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4021 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4081 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     4141 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     4201 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4261 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4321 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4381 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4441 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4501 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4561 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4621 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4681 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4741 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4801 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4861 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4921 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4981 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     5041 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5101 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5161 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5221 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5281 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5341 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5401 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5461 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     5521 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5581 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5641 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5701 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5761 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5821 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5881 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5941 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
