LOCUS       sequence-450597-        7340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Gluc-NHP.RMA and EGFP in the presence of Cre recombinase. 
            Double-floxed Gluc-NHP.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, Romac M, Watanabe S, Chernov M, Li H, Raisley E, Szablowski 
            HO, Costa VD
  TITLE     Synthetic Serum Markers Enable Noninvasive Monitoring of Gene 
            Expression in Primate Brains
  JOURNAL   bioRxiv 2025.06.01.657212; doi: 10.1101/2025.06.01.657212
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: "bioRxiv 
            2025.06.01.657212; doi: 10.1101/2025.06.01.657212"
COMMENT     Sequence Label: pAAV-hSyn-DIO-Gluc-NHP.RMA-IRES-EGFP
FEATURES             Location/Qualifiers
     source          1..7340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(660..693)
                     /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(744..777)
                     /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(786..1499,1500..1502,1503..1505))
                     /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(789..1499,1500..1502,1503..1505))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(831..852)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1180..1199
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1439..1460
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    join(1506..1514,1515..1517,1518..2092)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1679..1698)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     1908..1925
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(2758..3312)
                     /codon_start=1
                     /product="enhanced variant of secreted Gaussia luciferase"
                     /label=Gaussia-Dura Luc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGEKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIDDIPEIPGFKDLEPIEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGDD"
     CDS             complement(2758..3312)
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGEKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIDDIPEIPGFKDLEPIEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGDD"
     regulatory      3309..3318
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3325..3358
                     /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    3409..3442
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3467..4055
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3520..3540)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3938..3949)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4087..4563
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4286..4305)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4603..4743
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4603..4732
                     /label=AAV2 ITR
     rep_origin      4818..5273
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4905..4924)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5115..5136
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5290..5309)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5409..5431
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5469..5487)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5555..5659
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5660..5728,5729..6520)
                     /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(5878..5897)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6691..7279
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7180..7199
                     /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 gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgccctact tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc
      841 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc
      901 aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
      961 tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg
     1021 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
     1081 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
     1141 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
     1201 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     1261 tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc
     1321 acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc
     1381 ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
     1441 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     1501 accatggttg tggccatatt atcatcgtgt ttttcaaagg aaaaccacgt ccccgtggtt
     1561 cggggggcct agacgttttt ttaacctcga ctaaacacat gtaaagcatg tgtaccgagg
     1621 ccccagatca gatcccatac aatggggtac cttctgggca tccttcagcc ccttgttgaa
     1681 tacgcttgag gagagccatt tgactctttc cacaactatc caactcacaa cgtggcactg
     1741 gggttgtgcc gcctttgcag gtgtatctta tacacgtggc ttttggccgc agaggcacct
     1801 gtcgccaggt ggggggttcc gctgcctgca aagggtcgct acagacgttg tttgtcttca
     1861 agaagcttcc agaggaactg cttccttcac gacattcaac agaccttgca ttcctttggc
     1921 gagaggggaa agacccctag gaatgctcgt caagaagaca gggccaggtt tccgggccct
     1981 cacattgcca aaagacggca atatggtgga aaataacata tagacaaacg cacaccggcc
     2041 ttattccaag cggcttcggc cagtaacgtt aggggggggg gagggagagg ggcggatccc
     2101 ctagaatcat ttacccggag acacggagag gctcttctgg gtgtagtggt tgtgcagagc
     2161 ctcatgcatc acggagcatg agaagacgtt cccctgctgc cacctgctct tgtccacggt
     2221 gagcttgctg tagaggaagt aggagccgtc ggagtccagc acgggcggcg tggtcttgta
     2281 ggtgttctcc ggctgcccgc tgctctccca ctccacgacg atgtcgctgg ggtagaagcc
     2341 tttgaccagg caggtcaggc tgacctggtt cttggtcagc tcctcccggg acgggggcag
     2401 ggtgtacacc tgaggctctc ggggctgccc tttgtctttg gagatggttt tctggatggg
     2461 ggccgggagg gctttgttgg agaccttgca cgtgtactcc ttgccgttca gccagtcctg
     2521 gtgcgtgacg gtgaggacgc tgaccacacg atatgtgctg ttgtactgcg tctcccgtgg
     2581 cttcgtctgg gcatgatgca cctccgcgcc gtttacgtac cagttgaact tgacatcggg
     2641 gtcttcctgg ctcacgtcta ccaccacgca tgtgacctca ggggtccggg agatcatgag
     2701 ggtgtccttg ggttttgggg ggaagaggaa gactgacggt ccccccagga gggagccgtc
     2761 atcaccggcc cccttgatct tgtccacctg gccctggatc ttgctggcaa aggtcgcgca
     2821 gcgttgcggc agccacttct tgagcaggtc agaacactgc acgttggcaa gccctttgag
     2881 gcagccagtt gtgcagtcca cacacagatc gacctgtgcg atgaactgct ctatgggctc
     2941 caagtccttg aacccaggaa tctcaggaat gtcgtcgatc gcctcgccta tgccgccctg
     3001 tgcggactct ttgtcgcctt cgtaggtgtg gcagcgtcct gggatgaact tcttcatctt
     3061 gggcgtgcac ttgatgtggg acaggcagat cagacagccc ctggtgcagc cagctttccg
     3121 ggcattggct tccaactctt tgagcacctc cagcggcagc ttctcgccgg gcaacttccc
     3181 gcggtcagca tcgagatccg tggtcgcgaa gttgctggcc acggccacga tgttgaagtc
     3241 ttcgttgttc tcggtgggct tggcctcggc cacagcgatg cagatcaggg caaacagaac
     3301 tttgactccc atggtggcgc tagcataact tcgtataaag tatcctatac gaagttattt
     3361 gccttaaccc agaaattatc actgttattc tttagaatgg tgcaaagaat aacttcgtat
     3421 aatgtatgct atacgaagtt atgaattcga tatcaagctt atcgataatc aacctctgga
     3481 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     3541 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     3601 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     3661 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     3721 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     3781 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     3841 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct atgttgccac
     3901 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     3961 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     4021 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag
     4081 atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc
     4141 actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag
     4201 gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg
     4261 aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa
     4321 tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc
     4381 gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag
     4441 agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac
     4501 ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc
     4561 cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg
     4621 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     4681 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     4741 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     4801 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4861 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     4921 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4981 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     5041 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5101 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     5161 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     5221 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     5281 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     5341 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5401 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     5461 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     5521 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5581 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5641 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5701 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5761 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5821 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5881 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5941 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     6001 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     6061 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     6121 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6181 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     6241 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6301 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6361 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6421 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6481 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6541 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6601 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6661 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6721 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6781 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6841 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6901 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6961 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     7021 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     7081 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     7141 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     7201 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     7261 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     7321 ctggcctttt gctcacatgt
//
