LOCUS       Exported                6705 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  An AAV genome containing miRNA target sequences (TS) 204-5p and 122 
            to reduce expression of GCaMP6f in astrocytes and hepatocytes, 
            respectively.
ACCESSION   .
VERSION     .
KEYWORDS    CAG-GCaMP6f-3x-miR204-5p-3x-miR122-TS
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6705)
  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 6705)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6705
                     /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..2637
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1381..1390
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     1671..1692
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1798..1819)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2059..2078)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2832..3420
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2885..2905)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3303..3314)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3452..3928
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3651..3670)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3968..4108
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3968..4097
                     /label=AAV2 ITR
     rep_origin      4183..4638
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4270..4289)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4480..4501
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4655..4674)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4774..4796
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4834..4852)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4920..5024
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5025..5885
                     /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(5243..5262)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6056..6644
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6545..6564
                     /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 caccatgggt tctcatcatc atcatcatca tggtatggct
     1321 agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga taaggatctc
     1381 gccaccatgg tcgactcatc acgtcgtaag tggaataaga caggtcacgc agtcagagct
     1441 ataggtcggc tgagctcact cgagaacgtc tatatcaagg ccgacaagca gaagaacggc
     1501 atcaaggcga acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgcctac
     1561 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1621 ctgagcgtgc agtccaaact ttcgaaagac cccaacgaga agcgcgatca catggtcctg
     1681 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggcggt
     1741 accggaggga gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1801 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggtgagggc
     1861 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1921 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1981 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta catccaggag
     2041 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     2101 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     2161 atcctggggc acaagctgga gtacaacctg ccggaccaac tgactgaaga gcagatcgca
     2221 gaatttaaag aggaattctc cctatttgac aaggacgggg atgggacaat aacaaccaag
     2281 gagctgggga cggtgatgcg gtctctgggg cagaacccca cagaagcaga gctgcaggac
     2341 atgatcaatg aagtagatgc cgacggtgac ggcacaatcg acttccctga gttcctgaca
     2401 atgatggcaa gaaaaatgaa atacagggac acggaagaag aaattagaga agcgttcggt
     2461 gtgtttgata aggatggcaa tggctacatc agtgcagcag agcttcgcca cgtgatgaca
     2521 aaccttggag agaagttaac agatgaagag gttgatgaaa tgatcaggga agcagacatc
     2581 gatggggatg gtcaggtaaa ctacgaagag tttgtacaaa tgatgacagc gaagtgagaa
     2641 ttcaccggtc aaacaccatt gtcacactcc atactatcaa acaccattgt cacactccag
     2701 atcaacaaac accattgtca cactccacgc cggtaggcat aggatgacaa agggaatatc
     2761 acaggcatag gatgacaaag ggaactcaga aggcatagga tgacaaaggg aacgccggcg
     2821 aagctttcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     2881 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     2941 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     3001 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     3061 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3121 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3181 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     3241 cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3301 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3361 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     3421 atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc cctccccagt
     3481 gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt
     3541 aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt
     3601 ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga
     3661 accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca
     3721 agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct
     3781 cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc
     3841 caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc
     3901 gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt gcggaccgag
     3961 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     4021 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     4081 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     4141 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     4201 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4261 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4321 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4381 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4441 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     4501 gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     4561 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     4621 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4681 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4741 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4801 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     4861 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     4921 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4981 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5041 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5101 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5161 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5221 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5281 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5341 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5401 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5461 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5521 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5581 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5641 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5701 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5761 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5821 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5881 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5941 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6001 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6061 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6121 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6181 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     6241 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6301 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6361 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6421 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6481 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6541 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6601 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6661 cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
