LOCUS       AAV-CAG-fDIO-ArgiNLS-oScarlet        6213 bp ds-DNA     circular SYN 29-APR-2024
DEFINITION  An AAV genome with Flp recombinase-dependent expression of
            mNeonGreen from the CAG promoter.
ACCESSION
VERSION     .
KEYWORDS    pAAV-CAG-fDIO-mNeonGreen.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
REFERENCE   1  (bases 1 to 6156)
  AUTHORS   Chan KY, Jang MJ, Yoo BB, Greenbaum A, Ravi N, Wu WL,
            Sanchez-Guardado L, Lois C, Mazmanian SK, Deverman BE and Gradinaru
            V
  TITLE     Engineered AAVs for efficient noninvasive gene delivery to the
            central and peripheral nervous systems.
  JOURNAL   Nat Neurosci. 2017 Aug;20(8):1172-1179. doi: 10.1038/nn.4593. Epub
            2017 Jun 26.
  PUBMED    28671695
REFERENCE   2  (bases 1 to 6156)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
COMMENT     ##Imported_From-START##
            Path     :: /Users/ericszelenyi/Downloads
            Filename :: addgene-plasmid-99133-sequence-191157.gbk
            ##Imported_From-END##
FEATURES             Location/Qualifiers
     source          1..6213
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /note="Functional equivalent of wild-type AAV2 ITR"
                     /label="AAV2 ITR (alternate)"
     enhancer        210..513
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        515..792
                     /label="chicken beta-actin promoter"
     primer_bind     1122..1144
                     /note="Chimeric intron in CAG promoter, forward primer"
                     /label="pCAGGS-5"
     primer_bind     1206..1225
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
                     /label="pCAG-F"
     protein_bind    1292..1325
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
                     /label="FRT (minimal)"
     CDS             complement(1418..>2110)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved
                     from a synthetic template (<a
                     href=""https://www.ncbi.nlm.nih.gov/pubmed/27869816""
                     title=""https://www.ncbi.nlm.nih.gov/pubmed/27869816"">Bin
                     dels et al., 2016</a>)"
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWDRVMNFEDG
                     GAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGD
                     IKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSE
                     GRHSTGGMDELYK"
                     /transl_table=1
                     /modified_by="ericszelenyi"
                     /label="oScarlet"
     primer_bind     complement(1693..1712)
                     /Tm=54
                     /Sequence="CCCCGTAATGCAGAAGAAGA"
                     /Description="mCherry, forward primer"
                     /label="mCherry-F"
     primer_bind     1962..1980
                     /Tm=56
                     /Sequence="TTGGTCACCTTCAGCTTGG"
                     /Description="mCherry, reverse primer"
                     /label="mCherry-R"
     sig_peptide     complement(2111..2158)
                     /created_by="ericszelenyi"
                     /label="ArgiNLS"
     protein_bind    complement(2198..2231)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
                     /label="FRT (minimal)"
     misc_feature    2340..2928
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /label="WPRE"
     primer_bind     complement(2393..2413)
                     /note="WPRE, reverse primer"
                     /label="WPRE-R"
     CDS             complement(2811..2822)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
                     /label="Factor Xa site"
     polyA_signal    2960..3436
                     /note="human growth hormone polyadenylation signal"
                     /label="hGH poly(A) signal"
     primer_bind     complement(3159..3178)
                     /note="Human growth hormone terminator, reverse primer"
                     /label="hGH-PA-R"
     repeat_region   3476..3616
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /label="AAV2 ITR"
     repeat_region   3476..3605
                     /label="AAV2 ITR"
     rep_origin      3691..4146
                     /direction="RIGHT"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     primer_bind     complement(3778..3797)
                     /note="F1 origin, reverse primer"
                     /label="F1ori-R"
     primer_bind     3988..4009
                     /note="F1 origin, forward primer"
                     /label="F1ori-F"
     primer_bind     complement(4163..4182)
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /label="pRS-marker"
     primer_bind     4282..4304
                     /note="pGEX vectors, reverse primer"
                     /label="pGEX 3'"
     primer_bind     complement(4342..4360)
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
                     /label="pBRforEco"
     promoter        4428..4532
                     /gene="bla"
                     /label="AmpR promoter"
     CDS             4533..5393
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /label="AmpR"
     primer_bind     complement(4751..4770)
                     /note="Ampicillin resistance gene, reverse primer"
                     /label="Amp-R"
     rep_origin      5564..6152
                     /direction="RIGHT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     primer_bind     6053..6072
                     /note="pBR322 origin, forward primer"
                     /label="pBR322ori-F"
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 cgccaggcgg ggcggggcgg
      661 ggcgaggggc ggggcggggc 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 cggtaccgga tcctctagag tcgactccgg agaagttcct attctctaga aagtatagga
     1321 acttcgcaga atggtagctg gattgtagct gctattagca atatgaaacc tcttagaagt
     1381 tcctattctt caaaaggtat aggaacttcg gcgcgccctt gtacagctcg tccatgccgc
     1441 cggtggagtg gcggccctcg gagcgttcgt actgttccac cacggtgtag tcctcgttgt
     1501 gggaggtgat gtccaacttg cggtccacgt tgtaggcgcc gggcatctgc acgggcttct
     1561 tggccttgta ggtggtcttg aagtccgcca ggtagcgccc gccgtccttc aggcgcaggg
     1621 ccatcttaat gtcgcccttc agcacgccgt cctcggggta caaccgctcg gtggacgctt
     1681 cccagcccat tgtcttcttc tgcattacgg ggccgtcagg agggaagttg gtgccgcgga
     1741 gcttcacctt gtagatcagg gtgccgtcct ccagggaggt gtcctgggtc acggtcacgg
     1801 cgccgccgtc ctcgaagttc atcacgcggt cccacttgaa gccctcgggg aaggactgct
     1861 tatagtagtc ggggatgtcg gcggggtgct tggtgaaggc cctggagccg tacatgaact
     1921 gaggggacag gatgtcccag gagaagggca gggggccacc cttggtcacc ttcagcttgg
     1981 cggtctgggt gccctcgtag gggcggccct cgccctcgcc ctcgatctcg aactcgtggc
     2041 cgttcatgga gccctccatg tgcaccttga accgcatgaa ctccttgatc actgcctcgc
     2101 ccttgctcac tcgacggcgc cgccgccttc tcaccttctt tttctttgct atgctcatct
     2161 gcttctgggg agccaccctt ttcatggtgg cgctagcgaa gttcctatac tttctagaga
     2221 ataggaactt cttgccttaa cccagaaatt atcactgtta ttctttagaa tggtgcaaag
     2281 agaagttcct ataccttttg aagaatagga acttcgaatt cgatatcaag cttatcgata
     2341 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     2401 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     2461 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     2521 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     2581 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     2641 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     2701 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     2761 cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     2821 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     2881 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag
     2941 cgctgctcga gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc
     3001 cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat
     3061 tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa
     3121 ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag
     3181 tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct
     3241 gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt
     3301 gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc
     3361 tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc
     3421 tcccttccct gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa
     3481 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     3541 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     3601 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3661 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     3721 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     3781 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3841 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3901 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3961 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4021 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     4081 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     4141 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     4201 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4261 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4321 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     4381 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4441 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4501 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4561 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     4621 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     4681 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     4741 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     4801 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     4861 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     4921 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     4981 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5041 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5101 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5161 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5221 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     5281 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     5341 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     5401 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5461 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5521 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5581 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5641 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5701 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5761 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5821 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5881 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5941 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6001 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6061 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6121 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6181 tcctggcctt ttgctggcct tttgctcaca tgt
//
