LOCUS       sequence-356204-        6055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV genome with a CAG driven eGFP reporter with mR122 target 
            sequence repeats to reduce transgene expression in hepatocytes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6055)
  AUTHORS   Krolak T, Chan KY, Kaplan L, Huang Q, Wu J, Zheng Q, Kozareva V, 
            Beddow T, Tobey IG, Pacouret S, Chen AT, Chan YA, Ryvkin D, Gu C, 
            Deverman BE
  TITLE     A High-Efficiency AAV for Endothelial Cell Transduction Throughout 
            the Central Nervous System.
  JOURNAL   Nat Cardiovasc Res. 2022 Apr;1(4):389-400. doi: 
            10.1038/s44161-022-00046-4. Epub 2022 Apr 13.
  PUBMED    35571675
REFERENCE   2  (bases 1 to 6055)
  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"; doi: 
            "10.1038/s44161-022-00046-4"; journalName: "Nat Cardiovasc Res"; 
            date: "2022-04"; volume: "1"; issue: "4"; pages: "389-400"
COMMENT     Sequence Label: pAAV-CAG-SV40NLSf-GFP-3xmiR122-WPRE-HGHpA
FEATURES             Location/Qualifiers
     source          1..6055
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     CDS             1322..1342
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1352..1354,1355..1357,1358..2071)
                     /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             join(1352..1354,1355..1357,1358..2068)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1397..1418)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1658..1677)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2005..2026
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2182..2770
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2235..2255)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2653..2664)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2802..3278
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3001..3020)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3318..3458
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3318..3447
                     /label=AAV2 ITR
     rep_origin      3533..3988
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3620..3639)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3830..3851
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4005..4024)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4124..4146
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4184..4202)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4270..4374
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4375..4443,4444..5235)
                     /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(4593..4612)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5406..5994
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5895..5914
                     /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 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 cggtaccgcc accatgtcta gtgatgatga ggctactgct gactctcaac attctactcc
     1321 tccaaaaaag aagagaaagg tagaagaccc catggtgagc aagggcgagg agctgttcac
     1381 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1441 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1501 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1561 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1621 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1681 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1741 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1801 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1861 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1921 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     1981 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2041 cactctcggc atggacgagc tgtacaagta agaattcacc ggtcaaacac cattgtcaca
     2101 ctccatacta tcaaacacca ttgtcacact ccagatcaac aaacaccatt gtcacactcc
     2161 acgccggcga agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     2221 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     2281 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     2341 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     2401 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     2461 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     2521 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     2581 tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc
     2641 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     2701 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     2761 gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc
     2821 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc
     2881 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt
     2941 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
     3001 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct
     3061 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca
     3121 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca
     3181 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg
     3241 gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt
     3301 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     3361 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     3421 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     3481 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     3541 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3601 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3661 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3721 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     3781 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3841 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     3901 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3961 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     4021 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4081 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4141 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     4201 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4261 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4321 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4381 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4441 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4501 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4561 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4621 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4681 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4741 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4801 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4861 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     4921 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     4981 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5041 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5101 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5161 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5221 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5281 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5341 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5401 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5461 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5521 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     5581 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5641 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5701 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5761 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5821 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5881 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5941 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6001 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
