LOCUS       sequence-385082-        5893 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Astrocytic expression of Cre recombinase in AAV; astrocyte 
            specificity enhanced with 4x6T miRNA targeting cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5893)
  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 5893)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-GfaABC1D-Cre-4x6T
FEATURES             Location/Qualifiers
     source          1..5893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      23..611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     512..531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   673..802
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      1576..1585
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1582..2613
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MANLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     polyA_signal    3306..3782
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3505..3524)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3828..3968
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3828..3957
                     /label=AAV2 ITR
     rep_origin      4043..4498
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4130..4149)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4340..4361
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4515..4534)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4634..4656
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4694..4712)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4780..4884
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4885..4953,4954..5745)
                     /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"
ORIGIN
        1 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
       61 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
      121 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
      181 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
      241 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
      301 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
      361 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
      421 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
      481 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      541 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      601 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      661 ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt
      721 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      781 aactccatca ctaggggttc ctgcggccgc acgcgtacat atcctggtgt ggagtagggg
      841 acgctgctct gacagaggct cgggggcctg agctggctct gtgagctggg gaggaggcag
      901 acagccaggc cttgtctgca agcagacctg gcagcattgg gctggccgcc ccccagggcc
      961 tcctcttcat gcccagtgaa tgactcacct tggcacagac acaatgttcg gggtgggcac
     1021 agtgcctgct tcccgccgca ccccagcccc cctcaaatgc cttccgagaa gcccattgag
     1081 cagggggctt gcattgcacc ccagcctgac agcctggcat cttgggataa aagcagcaca
     1141 gccccctagg ggctgccctt gctgtgtggc gccaccggcg gtggagaaca aggctctatt
     1201 cagcctgtgc ccaggaaagg ggatcagggg atgcccaggc atggacagtg ggtggcaggg
     1261 ggggagagga gggctgtctg cttcccagaa gtccaaggac acaaatgggt gaggggagag
     1321 ctctccccat agctgggctg cggcccaacc ccaccccctc aggctatgcc agggggtgtt
     1381 gccaggggca cccgggcatc gccagtctag cccactcctt cataaagccc tcgcatccca
     1441 ggagcgagca gagccagagc aggttggaga ggagacgcat cacctccgct gctcgcttaa
     1501 ttaatacctg aattctgcag attcgagcgg ccgccagtgt gatggatatc tgcagaattc
     1561 aggtggccaa gatctgccac catggccaat ttactgaccg tacaccaaaa tttgcctgca
     1621 ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc tgatggacat gttcagggat
     1681 cgccaggcgt tttctgagca tacctggaaa atgcttctgt ccgtttgccg gtcgtgggcg
     1741 gcatggtgca agttgaataa ccggaaatgg tttcccgcag aacctgaaga tgttcgcgat
     1801 tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa ctatccagca acatttgggc
     1861 cagctaaaca tgcttcatcg tcggtccggg ctgccacgac caagtgacag caatgctgtt
     1921 tcactggtta tgcggcggat ccgaaaagaa aacgttgatg ccggtgaacg tgcaaaacag
     1981 gctctagcgt tcgaacgcac tgatttcgac caggttcgtt cactcatgga aaatagcgat
     2041 cgctgccagg atatacgtaa tctggcattt ctggggattg cttataacac cctgttacgt
     2101 atagccgaaa ttgccaggat cagggttaaa gatatctcac gtactgacgg tgggagaatg
     2161 ttaatccata ttggcagaac gaaaacgctg gttagcaccg caggtgtaga gaaggcactt
     2221 agcctggggg taactaaact ggtcgagcga tggatttccg tctctggtgt agctgatgat
     2281 ccgaataact acctgttttg ccgggtcaga aaaaatggtg ttgccgcgcc atctgccacc
     2341 agccagctat caactcgcgc cctggaaggg atttttgaag caactcatcg attgatttac
     2401 ggcgctaagg atgactctgg tcagagatac ctggcctggt ctggacacag tgcccgtgtc
     2461 ggagccgcgc gagatatggc ccgcgctgga gtttcaatac cggagatcat gcaagctggt
     2521 ggctggacca atgtaaatat tgtcatgaac tatatccgta acctggatag tgaaacaggg
     2581 gcaatggtgc gcctgctgga agatggcgat tagcatatgg gcattcaccg cgtgccttac
     2641 tacgcgtatt cttaagcaat aagcgaatat aacacggtcg atctaaaaag gttagctggg
     2701 tgtgttagaa gccctgcaag acgacctgcg cattattact cacggtacga cgcattatta
     2761 ctcacggtac gaggcattca ccgcgtgcct tactacgcgt attcttaagc aataagcgaa
     2821 tataacacgg tcgatctaaa aaggttagct gggtgtgtta gaagccctgc aagacgacct
     2881 gggcattcac cgcgtgcctt actacgcgta ttcttaagca ataagcgaat ataacacggt
     2941 cgatctaaaa aggttagctg ggtgtgttag aagccctgca agacgacctg ggtaccgcat
     3001 tattactcac ggtacgacgc attattactc acggtacgag gcattcaccg cgtgccttac
     3061 tacgcgtatt cttaagcaat aagcgaatat aacacggtcg atctaaaaag gttagctggg
     3121 tgtgttagaa gccctgcaag acgacctggg taccaggtaa agcggccgcg actctagatc
     3181 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     3241 cccctgaacc tgaaacataa aatgaatgca attctgcctc gagcagcgct gctcgagaga
     3301 tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca
     3361 ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg
     3421 tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga
     3481 agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat
     3541 cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg
     3601 agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga
     3661 gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc
     3721 caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc
     3781 ttctgatttt gtaggtaacc acgtggtggt gcggaccgag cggccgcagg aacccctagt
     3841 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     3901 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     3961 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4021 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4081 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     4141 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4201 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     4261 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4321 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     4381 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     4441 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     4501 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     4561 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4621 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4681 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     4741 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4801 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4861 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4921 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4981 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5041 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5101 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5161 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5221 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5281 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5341 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5401 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5461 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5521 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5581 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5641 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5701 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5761 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5821 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5881 gcgtcagacc ccg
//
