LOCUS       AAV-CAG-fDIO-ArgiNLS-mKate2        6216 bp ds-DNA     circular SYN 30-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/Desktop/oNLS_paper/PNAS_submission/
                        Addgene_Deposit
            Filename :: AAV-CAG-fDIO-ArgiNLS-mKate2.gb
            ##Imported_From-END##
FEATURES             Location/Qualifiers
     source          1..6216
                     /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..2113)
                     /product="<html><body>monomeric far-red fluorescent
                     protein (<a
                     href=""http://www.ncbi.nlm.nih.gov/pubmed/19143658""
                     title=""http://www.ncbi.nlm.nih.gov/pubmed/19143658"">Shch
                     erbo et al., 2009</a>)</body></html>"
                     /transl_table=1
                     /codon_start=1
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVL
                     TATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADM
                     ALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHR*"
                     /note="<html><body>mammalian
                     codon-optimized</body></html>"
                     /label="mKate2"
     sig_peptide     2114..2185
                     /created_by="TheGoldenLab"
                     /modified_by="ericszelenyi"
                     /label="ArgiNLS"
     protein_bind    complement(2201..2234)
                     /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    2343..2931
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /label="WPRE"
     primer_bind     complement(2396..2416)
                     /note="WPRE, reverse primer"
                     /label="WPRE-R"
     CDS             complement(2814..2825)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
                     /label="Factor Xa site"
     polyA_signal    2963..3439
                     /note="human growth hormone polyadenylation signal"
                     /label="hGH poly(A) signal"
     primer_bind     complement(3162..3181)
                     /note="Human growth hormone terminator, reverse primer"
                     /label="hGH-PA-R"
     repeat_region   3479..3619
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /label="AAV2 ITR"
     repeat_region   3479..3608
                     /label="AAV2 ITR"
     rep_origin      3694..4149
                     /direction="RIGHT"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     primer_bind     complement(3781..3800)
                     /note="F1 origin, reverse primer"
                     /label="F1ori-R"
     primer_bind     3991..4012
                     /note="F1 origin, forward primer"
                     /label="F1ori-F"
     primer_bind     complement(4166..4185)
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /label="pRS-marker"
     primer_bind     4285..4307
                     /note="pGEX vectors, reverse primer"
                     /label="pGEX 3'"
     primer_bind     complement(4345..4363)
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
                     /label="pBRforEco"
     promoter        4431..4535
                     /gene="bla"
                     /label="AmpR promoter"
     CDS             4536..5396
                     /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(4754..4773)
                     /note="Ampicillin resistance gene, reverse primer"
                     /label="Amp-R"
     rep_origin      5567..6155
                     /direction="RIGHT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     primer_bind     6056..6075
                     /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 gcgcgcctca tctgtgcccc agtttgctag
     1441 ggaggtcgca gtatctggcc acagccacct cgtgctgctc gacgtaggtc tctttgtcgg
     1501 cctccttgat tctttccagt cttctgtcca catagtagac gccgggcatc ttgaggttct
     1561 tagcgggttt cttggatctg tatgtggtct tcaagttgca gatcaggtgg cccccgccca
     1621 cgagcttcag ggccatgtcg gctctgcctt ccaggccgcc gtcagcgggg tacagggtct
     1681 cggtggaggc ctcccagccg agtgttttct tctgcatcac agggccgttg gatgggaagt
     1741 tcacccctct gatcttgacg ttgtagatga ggcagccgtc ctggaggctg gtgtcctggg
     1801 tagcggtcag cacgcccccg tcttcgtatg tggtgactct ctcccatgtg aagccctcgg
     1861 ggaaggactg cttaaagaag tcggggatgc cctgggtgtg gttgatgaag gttttgctgc
     1921 cgtacatgaa gctggtagcc aggatgtcga aggcgaaggg gagagggccg ccctcgaccg
     1981 ccttgattct catggtctgg gtgccctcgt agggcttgcc ttcgccctcg gatgtgcact
     2041 tgaagtggtg gttgttcacg gtgccctcca tgtacagctt catgtgcatg ttctccttaa
     2101 tcagctcgct cactcgacgg cgccgccgcc ttctcacctt ctttttcttt gctatgctca
     2161 tctgcttctg gggagccacc cttttcatgg tggcgctagc gaagttccta tactttctag
     2221 agaataggaa cttcttgcct taacccagaa attatcactg ttattcttta gaatggtgca
     2281 aagagaagtt cctatacctt ttgaagaata ggaacttcga attcgatatc aagcttatcg
     2341 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     2401 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     2461 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     2521 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     2581 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     2641 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     2701 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     2761 tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     2821 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     2881 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     2941 gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct
     3001 ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat
     3061 cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag
     3121 caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg
     3181 gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct
     3241 cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt
     3301 tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta
     3361 atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac
     3421 tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag
     3481 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     3541 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     3601 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3661 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     3721 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     3781 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3841 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3901 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3961 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     4021 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     4081 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     4141 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     4201 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4261 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4321 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     4381 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     4441 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     4501 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     4561 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     4621 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     4681 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     4741 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     4801 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     4861 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     4921 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     4981 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     5041 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     5101 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     5161 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     5221 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     5281 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     5341 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     5401 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     5461 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     5521 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5581 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5641 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5701 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5761 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5821 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5881 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5941 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6001 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6061 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6121 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6181 ggttcctggc cttttgctgg ccttttgctc acatgt
//
