LOCUS       sequence-385344-        6326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent AAV expression of GFP preferentially in astrocytes; 
            astrocyte selectivity generated with 4x6T miRNA targeting cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6326)
  AUTHORS   Gleichman AJ, Kawaguchi R, Sofroniew MV, Carmichael ST
  TITLE     A toolbox of astrocyte-specific, serotype-independent 
            adeno-associated viral vectors using microRNA targeting sequences.
  JOURNAL   Nat Commun. 2023 Nov 16;14(1):7426. doi: 10.1038/s41467-023-42746-w.
  PUBMED    37973910
REFERENCE   2  (bases 1 to 6326)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2023-11-16"; volume: "14(1):7426. doi"; pages: " 
            10.1038/s41467-023-42746-w"
COMMENT     Sequence Label: AAV-CAG-flex-GFP-4x6T
FEATURES             Location/Qualifiers
     source          1..6326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(19..607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(99..118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(778..1569,1570..1638))
                     /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     1401..1420
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1639..1743)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1769..2224)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1906..1927)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2118..2137
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2296..2425
                     /label=AAV2 ITR
     promoter        2736..3013
                     /label=chicken beta-actin promoter
     primer_bind     3292..3314
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3376..3395
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    3430..3463
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3526..3559
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(4144..4857,4858..4860,4861..4863))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(4147..4857,4858..4860,4861..4863))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4189..4210)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4538..4557
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4797..4818
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4860..4869
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(4886..4919)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(4990..5023)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5065..5653
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5118..5138)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5536..5547)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5696..5817
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5707..5726)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5761..5780
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5979..6108
                     /label=AAV2 ITR
ORIGIN
        1 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
       61 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      121 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      181 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
      241 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
      301 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
      361 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
      421 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
      481 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
      541 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
      601 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
      661 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
      721 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
      781 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
      841 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
      901 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
      961 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     1021 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     1081 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     1141 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     1201 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     1261 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     1321 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     1381 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     1441 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     1501 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     1561 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     1621 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     1681 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     1741 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     1801 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     1861 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     1921 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     1981 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     2041 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     2101 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     2161 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     2221 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     2281 tcgctattac gccagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg
     2341 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg
     2401 gccaactcca tcactagggg ttccttgtag ttaatgatta acccgccatg ctacttatct
     2461 acgtagccat gctctaggaa gagtaccatt gacgtcaata atgacgtatg ttcccatagt
     2521 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     2581 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     2641 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     2701 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
     2761 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
     2821 attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccrggsg gggsggggsg
     2881 gggsgrgggg sggggsgggg sgrggcggag aggkgcggcg gcagccaatc agagcggcgc
     2941 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
     3001 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc
     3061 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
     3121 cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct
     3181 gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc
     3241 ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct
     3301 tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt
     3361 tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat
     3421 tggatcccca taacttcgta taaagtatcc tatacgaagt tatatcaaaa taggaagacc
     3481 aatgcttcac catcgacccg aattgccaag catcaccatc gacccataac ttcgtataat
     3541 gtatgctata cgaagttata ctagggatcc ccccgtcgcg gccgctttac ctggtaccca
     3601 ggtcgtcttg cagggcttct aacacaccca gctaaccttt ttagatcgac cgtgttatat
     3661 tcgcttattg cttaagaata cgcgtagtaa ggcacgcggt gaatgcctcg taccgtgagt
     3721 aataatgcgt cgtaccgtga gtaataatgc ggtacccagg tcgtcttgca gggcttctaa
     3781 cacacccagc taaccttttt agatcgaccg tgttatattc gcttattgct taagaatacg
     3841 cgtagtaagg cacgcggtga atgcccaggt cgtcttgcag ggcttctaac acacccagct
     3901 aaccttttta gatcgaccgt gttatattcg cttattgctt aagaatacgc gtagtaaggc
     3961 acgcggtgaa tgcctcgtac cgtgagtaat aatgcgtcgt accgtgagta ataatgcgca
     4021 ggtcgtcttg cagggcttct aacacaccca gctaaccttt ttagatcgac cgtgttatat
     4081 tcgcttattg cttaagaata cgcgtagtaa ggcacgcggt gaatgcccat atgctaatcg
     4141 gttttacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca cgaactccag
     4201 caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact gggtgctcag
     4261 gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg
     4321 gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat
     4381 gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt tgtactccag
     4441 cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt
     4501 caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt
     4561 gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg
     4621 ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca gggtggtcac
     4681 gagggtgggc cagggcacgg gcagcttgcc ggtggtgcaa atgaacttca gggtcagctt
     4741 gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc
     4801 gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc ccttgctcac
     4861 catggtggcg gtaccgaatt ctagtataac ttcgtatagg atactttata cgaagttatc
     4921 attgggattc ttcctatttt gatccaagca tcaccatcga ccctctagtc cagatctcac
     4981 catcgaccca taacttcgta tagcatacat tatacgaagt tatgtccctc gaagaggttc
     5041 gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     5101 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     5161 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     5221 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     5281 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     5341 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     5401 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     5461 tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     5521 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     5581 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     5641 gccgcctccc cgcatcgata ccgtcgaccc gggcggccgc ttcgagcaga catgataaga
     5701 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     5761 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     5821 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa
     5881 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atcttcctag agcatggcta
     5941 cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt
     6001 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     6061 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcattaatga
     6121 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6181 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6241 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6301 cagcaaaagg ccaggaaccg taaaaa
//
