LOCUS       sequence-376528-        7340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Gluc-RMA and EGFP in the presence of Cre recombinase. 
            Double-floxed Gluc-RMA and EGFP for monitoring Cre-expressing 
            neuronal populations..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7340)
  AUTHORS   Lee S, Nouraein S, Kwon JJ, Huang Z, Wojick JA, Xia B, Corder G, 
            Szablowski JO
  TITLE     Engineered serum markers for non-invasive monitoring of gene 
            expression in the brain.
  JOURNAL   Nat Biotechnol. 2024 Jan 10. doi: 10.1038/s41587-023-02087-x.
  PUBMED    38200117
REFERENCE   2  (bases 1 to 7340)
  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 
            Biotechnol. 2024 Jan 10. doi: 10.1038/s41587-023-02087-x."
COMMENT     Sequence Label: pAAV-hSyn-DIO-Gluc-RMA-IRES-EGFP
FEATURES             Location/Qualifiers
     source          1..7340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(46..634)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(126..145)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(805..1596,1597..1665))
                     /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     1428..1447
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1666..1770)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1838..1856
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1894..1916)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2016..2035
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2052..2507)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2189..2210)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2401..2420
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2582..2722
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2593..2722
                     /label=AAV2 ITR
     polyA_signal    2762..3238
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     3020..3039
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    3270..3858
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3376..3387
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3785..3805
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(3883..3916)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(3967..4000)
                     /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)."
     regulatory      4007..4016
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4013..4567
                     /codon_start=1
                     /product="enhanced variant of secreted Gaussia luciferase"
                     /label=Gaussia-Dura Luc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGEKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIDDIPEIPGFKDLEPIEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGDD"
     CDS             4013..4567
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGEKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIDDIPEIPGFKDLEPIEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGDD"
     misc_feature    join(5233..5807,5808..5810,5811..5819)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5400..5417)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5627..5646
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(5820..5822,5823..5825,5826..6539)
                     /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(5820..5822,5823..5825,5826..6536)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5865..5886)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6126..6145)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6473..6494
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    6548..6581
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    6632..6665
                     /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)."
     promoter        complement(6703..7150)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     repeat_region   7195..7324
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
       61 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
      121 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
      181 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
      241 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
      301 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
      361 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
      421 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
      481 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
      541 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
      601 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
      661 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
      721 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
      781 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
      841 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
      901 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
      961 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     1021 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     1081 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     1141 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     1201 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     1261 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     1321 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     1381 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     1441 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     1501 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     1561 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     1621 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     1681 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     1741 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     1801 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     1861 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     1921 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     1981 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     2041 agtgcaccat aaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     2101 aatcagctca ttttttaacc aatagaccga aatcggcaaa atcccttata aatcaaaaga
     2161 atagcccgag atagagttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2221 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2281 accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2341 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2401 agggaagaaa gcgaaaggag cgggcgctaa ggcgctggca agtgtagcgg tcacgctgcg
     2461 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtact atggttgctt
     2521 tgacgtatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     2581 ccctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt
     2641 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
     2701 aactccatca ctaggggttc ctgcggccgc tcggtccgca cgtggttacc tacaaaatca
     2761 gaaggacagg gaagggagca gtggttcacg cctgtaatcc cagcaatttg ggaggccaag
     2821 gtgggtagat cacctgagat taggagttgg agaccagcct ggccaatatg gtgaaacccc
     2881 gtctctacca aaaaaacaaa aattagctga gcctggtcat gcatgcctgg aatcccaaca
     2941 actcgggagg ctgaggcagg agaatcgctt gaacccagga ggcggagatt gcagtgagcc
     3001 aagattgtgc cactgcactc cagcttggtt cccaatagac cccgcaggcc ctacaggttg
     3061 tcttcccaac ttgccccttg ctccatacca cccccctcca ccccataata ttatagaagg
     3121 acacctagtc agacaaaatg atgcaactta attttattag gacaaggctg gtgggcactg
     3181 gagtggcaac ttccagggcc aggagaggca ctggggaggg gtcacaggga tgccacccgt
     3241 agatctctcg agcagcgctc ggtatcgatg cggggaggcg gcccaaaggg agatccgact
     3301 cgtctgaggg cgaaggcgaa gacgcggaag aggccgcaga gccggcagca ggccgcggga
     3361 aggaaggtcc gctggattga gggccgaagg gacgtagcag aaggacgtcc cgcgcagaat
     3421 ccaggtggca acataggcga gcagccaagg aaaggacgat gatttccccg acaacaccac
     3481 ggaattgtca gtgcccaaca gccgagcccc tgtccagcag cgggcaaggc aggcggcgat
     3541 gagttccgcc gtggcaatag ggagggggaa agcgaaagtc ccggaaagga gctgacaggt
     3601 ggtggcaatg ccccaaccag tgggggttgc gtcagcaaac acagtgcaca ccacgccacg
     3661 ttgcctgaca acgggccaca actcctcata aagagacagc aaccaggatt tatacaagga
     3721 ggagaaaatg aaagccatac gggaagcaat agcatgatac aaaggcatta aagcagcgta
     3781 tccacatagc gtaaaaggag caacatagtt aagaatacca gtcaatcttt cacaaatttt
     3841 gtaatccaga ggttgattat cgataagctt gatatcgaat tcataacttc gtatagcata
     3901 cattatacga agttattctt tgcaccattc taaagaataa cagtgataat ttctgggtta
     3961 aggcaaataa cttcgtatag gatactttat acgaagttat gctagcgcca ccatgggagt
     4021 caaagttctg tttgccctga tctgcatcgc tgtggccgag gccaagccca ccgagaacaa
     4081 cgaagacttc aacatcgtgg ccgtggccag caacttcgcg accacggatc tcgatgctga
     4141 ccgcgggaag ttgcccggcg agaagctgcc gctggaggtg ctcaaagagt tggaagccaa
     4201 tgcccggaaa gctggctgca ccaggggctg tctgatctgc ctgtcccaca tcaagtgcac
     4261 gcccaagatg aagaagttca tcccaggacg ctgccacacc tacgaaggcg acaaagagtc
     4321 cgcacagggc ggcataggcg aggcgatcga cgacattcct gagattcctg ggttcaagga
     4381 cttggagccc atagagcagt tcatcgcaca ggtcgatctg tgtgtggact gcacaactgg
     4441 ctgcctcaaa gggcttgcca acgtgcagtg ttctgacctg ctcaagaagt ggctgccgca
     4501 acgctgcgcg acctttgcca gcaagatcca gggccaggtg gacaagatca agggggccgg
     4561 tgatgacggc tccgtcccag aagtatcatc tgtcttcatc ttccccccaa agcccaagga
     4621 tgtgctcacc attactctga ctcctaaggt cacgtgtgtt gtggtagaca tcagcaagga
     4681 tgatcccgag gtccagttca gctggtttgt agatgatgtg gaggtgcaca cagctcagac
     4741 gcaaccccgg gaggagcagt tcaacagcac tttccgctca gtcagtgaac ttcccatcat
     4801 gcaccaggac tggctcaatg gcaaggagtt caaatgcagg gtcaacagtg cagctttccc
     4861 tgcccccatc gagaaaacca tctccaaaac caaaggcaga ccgaaggctc cacaggtgta
     4921 caccattcca cctcccaagg agcagatggc caaggataaa gtcagtctga cctgcatgat
     4981 aacagacttc ttccctgaag acattactgt ggagtggcag tggaatgggc agccagcgga
     5041 gaactacaag aacactcagc ccatcatgga cacagatggc tcttacttcg tctacagcaa
     5101 gctcaatgtg cagaagagca actgggaggc aggaaatact ttcacctgct ctgtgttaca
     5161 tgagggcctg cacaaccacc atactgagaa gagcctctcc cactctcctg gtaaatgatt
     5221 ctaggggatc cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga
     5281 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
     5341 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     5401 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     5461 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     5521 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     5581 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     5641 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
     5701 ggggcctcgg tacacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc
     5761 cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacca
     5821 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     5881 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     5941 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     6001 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     6061 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     6121 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     6181 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     6241 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     6301 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     6361 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     6421 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     6481 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtagg
     6541 gcgcgccata acttcgtata atgtatgcta tacgaagtta ttaagaggtt tcatattgct
     6601 aatagcagct acaatccagc taccattctg cataacttcg tataaagtat cctatacgaa
     6661 gttattccgg agtcgactct agaggatccg gtaccttctc gactgcgctc tcaggcacga
     6721 cacgactcct ccgctgccca ccgcagactg aggcagcgct gagtcgccgg cgccgcagcg
     6781 cagatggtcg cgcccgtgcc cccctatctc gcgcctcgcg tggtgcggtc cggctgggcc
     6841 ggcggcggcg cggacgcgac caaggtggcc gggaagggga gtttgcgggg gaccggcgag
     6901 tgacgtcagc gcgccttcag tgctgaggcg gcggtggcgc gcgccgccag gcgggggcga
     6961 aggcactgtc cgcggtgctg aagctggcag tgcgcacgcg cctcgccgca tcctgtttcc
     7021 cctccccctc tctgataggg gatgcgcaat ttggggaatg ggggttgggt gcttgtccag
     7081 tgggtcgggg tcggtcgtca ggtaggcacc cccaccccgc ctcatcctgg tcctaaaacc
     7141 cacttgcact catacgcagg gccctctgca gtctagacac acgcgtgcgg ccgcaggaac
     7201 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     7261 gaccaaaggt cgcccgacgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     7321 caggacatgt gagcaaaagg
//
