LOCUS       Exported                6695 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 6695)
  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 6695)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6695
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        210..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        515..792
                     /label=chicken beta-actin promoter
     primer_bind     1122..1144
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1206..1225
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1268..1277
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1274..2626
                     /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      1370..1379
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     1660..1681
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1787..1808)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2048..2067)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2822..3410
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2875..2895)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3293..3304)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3442..3918
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3641..3660)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3958..4098
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3958..4087
                     /label=AAV2 ITR
     rep_origin      4173..4628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4260..4279)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4470..4491
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4645..4664)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4764..4786
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4824..4842)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4910..5014
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5015..5875
                     /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(5233..5252)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6046..6634
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6535..6554
                     /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 gcgtaacact agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
      541 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
      601 ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccrggcgg ggcggggsgg
      661 ggsgrggggs ggggsggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
      721 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
      781 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
      841 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
      901 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg
      961 tggctgcgtg aaagccttga ggggctccgg gagcgccggc aggaaggaaa tgggcgggga
     1021 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1081 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1141 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1201 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgcggcccaa
     1261 cggtaccgcc accatgggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
     1321 tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatctcg ccaccatggt
     1381 cgactcatca cgtcgtaagt ggaataagac aggtcacgca gtcagagcta taggtcggct
     1441 gagctcactc gagaacgtct atatcaaggc cgacaagcag aagaacggca tcaaggcgaa
     1501 cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc actaccagca
     1561 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcgtgca
     1621 gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1681 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggta ccggagggag
     1741 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1801 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggtgagggcg atgccaccta
     1861 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1921 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1981 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac atccaggagc gcaccatctt
     2041 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2101 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2161 caagctggag tacaacctgc cggaccaact gactgaagag cagatcgcag aatttaaaga
     2221 ggaattctcc ctatttgaca aggacgggga tgggacaata acaaccaagg agctggggac
     2281 ggtgatgcgg tctctggggc agaaccccac agaagcagag ctgcaggaca tgatcaatga
     2341 agtagatgcc gacggtgacg gcacaatcga cttccctgag ttcctgacaa tgatggcaag
     2401 aaaaatgaaa tacagggaca cggaagaaga aattagagaa gcgttcggtg tgtttgataa
     2461 ggatggcaat ggctacatca gtgcagcaga gcttcgccac gtgatgacaa accttggaga
     2521 gaagttaaca gatgaagagg ttgatgaaat gatcagggaa gcagacatcg atggggatgg
     2581 tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg aagtgagaat tcaccggtca
     2641 aacaccattg tcacactcca tactatcaaa caccattgtc acactccaga tcaacaaaca
     2701 ccattgtcac actccacgcc ggtaggcata ggatgacaaa gggaatatca caggcatagg
     2761 atgacaaagg gaactcagaa ggcataggat gacaaaggga acgccggcga agcttatcga
     2821 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     2881 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     2941 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     3001 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     3061 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     3121 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     3181 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     3241 cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     3301 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     3361 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     3421 agcgctgctc gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg
     3481 gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc
     3541 attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc
     3601 aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg
     3661 agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc
     3721 ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt
     3781 ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa
     3841 tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact
     3901 gctcccttcc ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg
     3961 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     4021 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     4081 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4141 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     4201 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     4261 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4321 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4381 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4441 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4501 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     4561 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     4621 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     4681 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4741 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4801 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     4861 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4921 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4981 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5041 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5101 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5161 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5221 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5281 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5341 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5401 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5461 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5521 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5581 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5641 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5701 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5761 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5821 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     5881 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5941 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6001 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6061 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6121 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6181 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6241 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6301 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6361 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6421 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6481 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6541 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6601 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6661 gttcctggcc ttttgctggc cttttgctca catgt
//
