LOCUS       sequence-275771-        7313 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express hChR2(H134R)-P2A-EYFP in a Cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7313)
  AUTHORS   Chen W, Cai ZL, Chao ES, Chen H, Longley CM, Hao S, Chao HT, Kim JH,
            Messier JE, Zoghbi HY, Tang J, Swann JW, Xue M
  TITLE     Stxbp1/Munc18-1 haploinsufficiency impairs inhibition and mediates 
            key neurological features of STXBP1 encephalopathy.
  JOURNAL   Elife. 2020 Feb 19;9. pii: 48705. doi: 10.7554/eLife.48705.
  PUBMED    32073399
REFERENCE   2  (bases 1 to 7313)
  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: "Elife."; 
            date: "2020-02-19"; volume: "9. pii: 48705. doi"; pages: " 
            10.7554/eLife.48705"
COMMENT     Sequence Label: pAAV-EF1a-DIO-hChR2(H134R)-P2A-EYFP
FEATURES             Location/Qualifiers
     source          1..7313
                     /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        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1440..1473)
                     /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(1524..1557)
                     /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(1566..2279,2280..2282))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1611..1632)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1960..1979
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2219..2240
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2283..2339)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(2361..3287)
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      3284..3293
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3298..3331
                     /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    3382..3415
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3440..4028
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3493..3513)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3911..3922)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4060..4536
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4259..4278)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4576..4716
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4576..4705
                     /label=AAV2 ITR
     rep_origin      4791..5246
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4878..4897)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5088..5109
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5263..5282)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5382..5404
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5442..5460)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5528..5632
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5633..5701,5702..6493)
                     /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(5851..5870)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6664..7252
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7153..7172
                     /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgccttact tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc
     1621 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctggtagctc
     1681 aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
     1741 tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg
     1801 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
     1861 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
     1921 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
     1981 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     2041 tgctgcttca tgtggtcggg gtagcgggcg aagcactgca ggccgtagcc gaaggtggtc
     2101 acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc
     2161 ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
     2221 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     2281 acgggaccgg ggttttcttc cacgtctcct gcttgcttta acagagagaa gttcgtggct
     2341 ccggagccgg gcgcgcctgg cacggctccg gcctcggctt cgtcttcgac gagagtctcg
     2401 acctcgatct ccgttccgcc gatgttcagt ttggtggttt tgcggatatc tccgtgaatc
     2461 aatatgtgct cgtggatcag gacgcgcagg tagtgtccca acaaccccca acaattttta
     2521 ctcatcagat caataatcgt gtgacctacg gtggagccat agacgctcag gacgccaaaa
     2581 ccttcgggcc ccaaaatgaa gagaattggg aacatacccc agctcacgaa aaacagccat
     2641 gccatgccgg tcacgacctg gcggcaccga ccctttggca cagtatgata accctcgata
     2701 tatgctttgg cggcgtgaaa aaatgtgttc gcgccatagc acaatccaag acaaaagaag
     2761 atgactttaa catagccggt tgccatggcg ctggtagccc cccacacgat agtcccgatg
     2821 tctgagacaa ggagtcccat ggttctcctg ctgtagtcgt tgctcaggcc ggtgaggttg
     2881 ctcaggcgga taaggatgac aggacaagtg agcagccact ctgcatagcg cagccactgc
     2941 acccggtgtc ctgtggcaag gtagagcata gagggattct taaactcaaa aaagaactcg
     3001 agaatcacct taaccatttc aatggcgcac acatagatct cctcccagcc gcatgtagat
     3061 ttccaggttt ggtaggcata gaacatcagc agcaaaatgc tgaatcctgc tgcaagccac
     3121 tgcaggacat ttgacgcggt ctgagcgccg ttcgtgccgc gagattcaat ccatccggca
     3181 cagtaacatt gatcctcagg gaccaggacg gacccgttca ccaccacagg attagtaacg
     3241 aacaaaagtt cgcgtccgac ggcagacaaa gcgccgccat agtccatggt ggctagcata
     3301 acttcgtata aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta
     3361 ttctttagaa tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt
     3421 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3481 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3541 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3601 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3661 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3721 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3781 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     3841 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     3901 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     3961 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4021 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc
     4081 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
     4141 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
     4201 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
     4261 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
     4321 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
     4381 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
     4441 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
     4501 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc
     4561 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4621 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4681 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4741 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4801 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4861 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4921 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4981 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     5041 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5101 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     5161 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5221 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     5281 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5341 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5401 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     5461 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5521 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5581 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5641 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5701 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5761 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5821 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5881 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5941 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6001 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6061 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6121 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6181 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6241 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6301 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6361 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6421 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6481 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6541 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6601 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6661 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6721 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6781 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6841 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6901 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6961 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7021 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     7081 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7141 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7201 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7261 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
