LOCUS       sequence-481862-        7436 bp    DNA     circular SYN 04-AUG-2025
DEFINITION  The transfer plasmid for packaging AAV expressing Cre-dependent 
            chicken OPN5 was constructed by inserting the OPN5 coding sequence 
            into the multiple cloning site under the control of EF1-alpha 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7436)
  AUTHORS   Dai R, Yu T, Weng D, Li H, Cui Y, Wu Z, Guo Q, Zou H, Wu W, Gao X, 
            Qi Z, Ren Y, Wang S, Li Y, Luo M
  TITLE     A neuropsin-based optogenetic tool for precise control of Gq 
            signaling.
  JOURNAL   Sci China Life Sci. 2022 Jul;65(7):1271-1284. doi: 
            10.1007/s11427-022-2122-0. Epub 2022 May 12.
  PUBMED    35579776
REFERENCE   2  (bases 1 to 7436)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1007/s11427-022-2122-0"; journalName: "Sci China Life Sci"; 
            date: "2022-07"; volume: "65"; issue: "7"; pages: "1271-1284"
COMMENT     Sequence Label: pAAV-EF1-alpha-DIO-cOpn5-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..7436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        881..2059
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1112..2050
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2007..2027
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(2090..2123)
                     /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(2174..2207)
                     /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(2219..2932,2933..2935))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(2222..2932,2933..2935))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2264..2285)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2613..2632
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2872..2893
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2936..2989)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    4071..4104
                     /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    4155..4188
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4213..4801
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4266..4286)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4684..4695)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4833..5309
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5032..5051)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5349..5489
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5349..5478
                     /label=AAV2 ITR
     rep_origin      5564..6019
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5651..5670)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5861..5882
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6036..6055)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6155..6177
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6215..6233)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6301..6405
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6406..6474,6475..7266)
                     /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(6624..6643)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga ggaatctttg
      841 cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg
      901 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
      961 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     1021 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     1081 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc
     1141 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta
     1201 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc
     1261 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
     1321 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
     1381 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
     1441 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
     1501 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
     1561 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     1621 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     1681 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg
     1741 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     1801 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag
     1861 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     1921 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     1981 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     2041 tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa taacttcgta
     2101 taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc tattagcaat
     2161 atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc gcgccttatt
     2221 acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     2281 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt
     2341 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg
     2401 cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt
     2461 tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt
     2521 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     2581 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     2641 agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     2701 tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg
     2761 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt
     2821 aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     2881 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcactgggc
     2941 caggattctc ctcgacgtca ccgcatgtta gcagacttcc tctgccctcg acttccagtt
     3001 gggttccgct cagtgccgct gaatctctgt gtgaagataa ggtgtacatc ctcgattcct
     3061 tcaaagagct cttcttctgc agtgtcttcg gcccacctga ccggcagcag gcaaatttgc
     3121 aatcaatgac ctggtaaata attgggttgt acatggcagc cgacttggca agcagagttg
     3181 gtaccaccga aaattgaatg ggaacggaat ccggctgtcc aaaggctgac cacacagaga
     3241 ccacggcgta ggggatccaa gcaatgagga atccggcaca gatcagcatt gccaccttgg
     3301 tcagcttcat ttcaagtatg tggctgttct ggatcctggt gtcatagtga gcaacttctt
     3361 tggtggacga cttgactttt aaaataattt taacatagga gaacacaatc actgcagttg
     3421 gaaacaggag gcaaaagaaa agaatgctga gaacaaaagc ctgcccagcc actgaagcct
     3481 gcgccagcca ccagtccagg gtgcacgagg tcccaaacgg ctcaggagcg tagctcccca
     3541 caccagcaaa aggcactgta gcccagaacg tcgcataggc ccagattagt gccaggcaga
     3601 taaaggcatg gtgtctctta agccaggtac cataagccaa gtgacagatt tttaagtatc
     3661 tatccaggct gacggctgtc atggtaataa ggctcccaca gccaaagaag aagccagccc
     3721 atccgtacca gcggcagccc atccatccaa agatccagcg gtgagaaaag aaggaaatga
     3781 tactgaaggg ttttcctaca actgagatgc ccagatcaca caccgctaaa ttaacagtca
     3841 ttatctcagc gggtcttaac ttctttttcc gctttgagga cataaagata acatacccgt
     3901 ttcccagagt tgaaagaatc ccaattactg tcaaataaaa ccccgctatg atgtcagctt
     3961 ctctagacag ctttgatgca aaggggtctt cctgctgcac gtagtgtggg aggtattctt
     4021 cctgagagga ggagttgcag tccgatgcca tcccactcat ggtggctagc ataacttcgt
     4081 ataaagtatc ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta
     4141 gaatggtgca aagaataact tcgtataatg tatgctatac gaagttatga attcgatatc
     4201 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     4261 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     4321 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     4381 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     4441 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     4501 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     4561 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     4621 ccttggctgc tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     4681 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     4741 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     4801 catcgatacc gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag
     4861 tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat
     4921 taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg
     4981 tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg
     5041 aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc
     5101 aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc
     5161 tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct
     5221 ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg
     5281 cgtgaaccac tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga
     5341 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     5401 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     5461 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     5521 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     5581 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5641 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5701 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5761 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5821 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5881 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     5941 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     6001 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     6061 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     6121 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     6181 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     6241 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     6301 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     6361 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     6421 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     6481 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     6541 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     6601 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     6661 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6721 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6781 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6841 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6901 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6961 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     7021 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     7081 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     7141 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     7201 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     7261 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     7321 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     7381 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
