LOCUS       sequence-451474-        7581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV construct for Cre-dependent (FLEX) expression of macaque 
            melanopsin (Opn4, short isoform, GenBank:OP856641; hSyn promoter) 
            and of EGFP via an IRES..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7581)
  AUTHORS   Liu A, Milner ES, Peng YR, Blume HA, Brown MC, Bryman GS, Emanuel 
            AJ, Morquette P, Viet NM, Sanes JR, Gamlin PD, Do MTH
  TITLE     Encoding of environmental illumination by primate melanopsin 
            neurons.
  JOURNAL   Science. 2023 Jan 27;379(6630):376-381. doi: 
            10.1126/science.ade2024. Epub 2023 Jan 26.
  PUBMED    36701440
REFERENCE   2  (bases 1 to 7581)
  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: 
            "10.1126/science.ade2024"; journalName: "Science"; date: 
            "2023-01-27- 27"; volume: "379"; issue: "6630"; pages: "376-381"
COMMENT     Sequence Label: AAV-hSyn-FLEX-macaque OPN4-IRES-EGFP
FEATURES             Location/Qualifiers
     source          1..7581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(974..1548,1549..1551,1552..1560)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1141..1158)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1368..1387
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1561..1563,1564..1566,1567..2280)
                     /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(1561..1563,1564..1566,1567..2277)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1606..1627)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1867..1886)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2214..2235
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2289..2322
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2373..2406
                     /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(2444..2891)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     repeat_region   2936..3065
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(3127..3715)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3207..3226)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3886..4677,4678..4746))
                     /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     4509..4528
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4747..4851)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4919..4937
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4975..4997)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5097..5116
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5133..5588)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5270..5291)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5482..5501
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5663..5803
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5674..5803
                     /label=AAV2 ITR
     polyA_signal    5843..6319
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     6101..6120
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    6351..6939
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6457..6468
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     6866..6886
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(6964..6997)
                     /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(7048..7081)
                     /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)."
ORIGIN
        1 cctggaccgc tacctggtaa tcacacgccc gctggccacc attggtgtgg cgtccaagag
       61 gcgcgcggca tttgtcctgc tgggcgtttg gctctatgcc ctggcctgga gtctgccacc
      121 cttctttggc tggagtgcct atgtgcccga ggggttgctg acatcctgct cctgggacta
      181 catgagcttc acaccggctg tgcgtgccta caccatgctt ctctgctgct tcgtgttctt
      241 cctccctctg cttatcatca tctactgcta catcttcatc ttcagggcca tccgggagac
      301 aggacaggct ctccagacct tcggggcctg caagggcagt ggcgagtccc tgtggcagcg
      361 gcagcggctg cagagcgagt gcaagatggc caagatcatg ctgctggtca tcctcctctt
      421 cgtgctctcc tgggctccct attctgctgt ggccctggtg gcctttgctg ggtacgcaca
      481 tgtcctgaca ccctacatga gctcggtgcc agccgtcatc gccaaggcct ctgcaatcca
      541 caaccccatc atttacgcca tcacccaccc caagtacagg gtggccattg cccagcacct
      601 gccctgcctg ggggtgctgc tgggtgtatc acgccggcac agtcacccct accccagcta
      661 ccgctccacc caccgctcca cgctgatcag ccatacctcc aacctcagct ggatctctgg
      721 acggaggcgc caggagtccc tgggctcaga gagtgaggtg ggctggacac acatggaggc
      781 agcagctgtg tggggagccg cccagcaagc gaatgggcgg tccctctacg gtcagggtct
      841 ggaggacttg gaagccaagg cgccccccag accccaggga caggaagcag agactccagg
      901 gaagaccaag gggctgctcc cctgcaagga ctccaggatg taggcggccg ctacgtaaat
      961 tccgcccccc ccccccctct ccctcccccc cccctaacgt tactggccga agccgcttgg
     1021 aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca
     1081 atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc
     1141 ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag
     1201 cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg
     1261 gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac
     1321 aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa
     1381 gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc
     1441 tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc
     1501 ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc
     1561 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1621 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1681 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1741 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1801 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1861 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1921 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1981 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2041 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2101 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2161 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2221 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     2281 ggcgcgccat aacttcgtat aatgtatgct atacgaagtt attaagaggt ttcatattgc
     2341 taatagcagc tacaatccag ctaccattct gcataacttc gtataaagta tcctatacga
     2401 agttattccg gagtcgactc tagaggatcc ggtaccttct cgactgcgct ctcaggcacg
     2461 acacgactcc tccgctgccc accgcagact gaggcagcgc tgagtcgccg gcgccgcagc
     2521 gcagatggtc gcgcccgtgc ccccctatct cgcgcctcgc gtggtgcggt ccggctgggc
     2581 cggcggcggc gcggacgcga ccaaggtggc cgggaagggg agtttgcggg ggaccggcga
     2641 gtgacgtcag cgcgccttca gtgctgaggc ggcggtggcg cgcgccgcca ggcgggggcg
     2701 aaggcactgt ccgcggtgct gaagctggca gtgcgcacgc gcctcgccgc atcctgtttc
     2761 ccctccccct ctctgatagg ggatgcgcaa tttggggaat gggggttggg tgcttgtcca
     2821 gtgggtcggg gtcggtcgtc aggtaggcac ccccaccccg cctcatcctg gtcctaaaac
     2881 ccacttgcac tcatacgcag ggccctctgc agtctagaca cacgcgtgcg gccgcaggaa
     2941 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     3001 cgaccaaagg tcgcccgacg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     3061 gcaggacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3121 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3181 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3241 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3301 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3361 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3421 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3481 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3541 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3601 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3661 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3721 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3781 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3841 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3901 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3961 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4021 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4081 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4141 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4201 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4261 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4321 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4381 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4441 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4501 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4561 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4621 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4681 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4741 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4801 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4861 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     4921 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     4981 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     5041 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     5101 atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata ttttgttaaa
     5161 attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg aaatcggcaa
     5221 aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc cagtttggaa
     5281 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     5341 gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg
     5401 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     5461 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta aggcgctggc
     5521 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     5581 gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag atgcgtaagg
     5641 agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg ctcgctcgct cactgaggcc
     5701 gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
     5761 gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg ctcggtccgc
     5821 acgtggttac ctacaaaatc agaaggacag ggaagggagc agtggttcac gcctgtaatc
     5881 ccagcaattt gggaggccaa ggtgggtaga tcacctgaga ttaggagttg gagaccagcc
     5941 tggccaatat ggtgaaaccc cgtctctacc aaaaaaacaa aaattagctg agcctggtca
     6001 tgcatgcctg gaatcccaac aactcgggag gctgaggcag gagaatcgct tgaacccagg
     6061 aggcggagat tgcagtgagc caagattgtg ccactgcact ccagcttggt tcccaataga
     6121 ccccgcaggc cctacaggtt gtcttcccaa cttgcccctt gctccatacc acccccctcc
     6181 accccataat attatagaag gacacctagt cagacaaaat gatgcaactt aattttatta
     6241 ggacaaggct ggtgggcact ggagtggcaa cttccagggc caggagaggc actggggagg
     6301 ggtcacaggg atgccacccg tagatctctc gagcagcgct cggtatcgat gcggggaggc
     6361 ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga agacgcggaa gaggccgcag
     6421 agccggcagc aggccgcggg aaggaaggtc cgctggattg agggccgaag ggacgtagca
     6481 gaaggacgtc ccgcgcagaa tccaggtggc aacataggcg agcagccaag gaaaggacga
     6541 tgatttcccc gacaacacca cggaattgtc agtgcccaac agccgagccc ctgtccagca
     6601 gcgggcaagg caggcggcga tgagttccgc cgtggcaata gggaggggga aagcgaaagt
     6661 cccggaaagg agctgacagg tggtggcaat gccccaacca gtgggggttg cgtcagcaaa
     6721 cacagtgcac accacgccac gttgcctgac aacgggccac aactcctcat aaagagacag
     6781 caaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa tagcatgata
     6841 caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt taagaatacc
     6901 agtcaatctt tcacaaattt tgtaatccag aggttgatta tcgataagct tgatatcgaa
     6961 ttcataactt cgtatagcat acattatacg aagttattct ttgcaccatt ctaaagaata
     7021 acagtgataa tttctgggtt aaggcaaata acttcgtata ggatacttta tacgaagtta
     7081 tgctagcatg aaccctcctt cgggaccaag agtcccgccg agcccaaccc aggagcccag
     7141 ctgcatggcc accgcagccc cacccagcag gtgggacagc tcccagagca gcatctccag
     7201 cctgggccag cttccgtccg tcagtcccac ggcagctggg acttgggctg ctgcctgggt
     7261 ccccttcccc acggttgatg ttccagacca tgcccactat actctgggca cagtgatctt
     7321 gctggtggga ctcacgggga tgctgggcaa cctgacggtc atctatactt tctgcaggag
     7381 cagaggcctc cggacacctg ccaacatgtt cattatcaac ctcgccatca gcgacttcct
     7441 catgtccttc acccaggccc ctgtcttctt cgccagcagc ctctataagc actggctctt
     7501 tggggagaca ggctgcgagt tctatgcctt ctgtggggct ctctttggca tctcctccat
     7561 gatcaccctg acggccatcg c
//
