LOCUS       Exported                5994 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  An AAV genome containing miRNA target sequence (TS) 204-5p to reduce
            expression of the fluorescent protein eYFP in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    CAG-eYFP-3x-miR204-5p-TS
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5994)
  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 5994)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5994
                     /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      1286..1295
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1292..2011
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1337..1358)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1598..1617)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1945..1966
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2121..2709
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2174..2194)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2592..2603)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2741..3217
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2940..2959)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3257..3397
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3257..3386
                     /label=AAV2 ITR
     rep_origin      3472..3927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3559..3578)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3769..3790
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3944..3963)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4063..4085
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4123..4141)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4209..4313
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4314..5174
                     /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(4532..4551)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5345..5933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5834..5853
                     /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 cacccgccac catggtgagc aagggcgagg agctgttcac
     1321 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1381 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1441 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgca
     1501 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1561 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1621 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1681 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1741 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1801 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1861 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc taccagtccg ccctgagcaa
     1921 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1981 cactctcggc atggacgagc tgtacaagtg agaattcacc ggtaggcata ggatgacaaa
     2041 gggaatatca caggcatagg atgacaaagg gaactcagaa ggcataggat gacaaaggga
     2101 acgccggcga agctttcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     2161 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     2221 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     2281 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     2341 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     2401 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     2461 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     2521 cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct
     2581 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     2641 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     2701 cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc cctgtgaccc
     2761 ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct
     2821 aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg
     2881 gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt
     2941 ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc
     3001 ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat
     3061 gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag
     3121 gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg
     3181 attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccacgtg
     3241 cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     3301 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     3361 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     3421 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     3481 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3541 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3601 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3661 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     3721 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3781 tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt
     3841 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3901 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     3961 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4021 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4081 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     4141 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     4201 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4261 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4321 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4381 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4441 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4501 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4561 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4621 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4681 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4741 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4801 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     4861 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4921 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4981 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5041 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5101 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5161 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5221 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5281 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5341 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5401 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5461 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     5521 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5581 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5641 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5701 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5761 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5821 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5881 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5941 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
