LOCUS       sequence-9545-ex        7266 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-activated AAV expression of humanized ChR2 with H134R mutation 
            fused to EYFP for optogenetic activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7266)
  TITLE     Cre-activated AAV expression plasmids
REFERENCE   2  (bases 1 to 7266)
  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"
COMMENT     Sequence Label: pAAV-EF1a-double floxed-hChR2(H134R)-EYFP-WPRE...
FEATURES             Location/Qualifiers
     source          1..7266
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1450..1483)
                     /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(1534..1567)
                     /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(1579..2292,2293..2295,2296..2298))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1624..1645)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1973..1992
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2232..2253
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2295..2304
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2314..3240)
                     /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      3237..3246
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3251..3284
                     /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    3335..3368
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3393..3981
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3446..3466)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3864..3875)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4013..4489
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4212..4231)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4529..4669
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4529..4658
                     /label=AAV2 ITR
     rep_origin      4744..5199
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4831..4850)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5041..5062
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5216..5235)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5335..5357
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5395..5413)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5481..5585
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5586..5654,5655..6446)
                     /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(5804..5823)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6617..7205
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7106..7125
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1441 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1561 aagttatggc gcgccctatt acttgtacag ctcgtccatg ccgagagtga tcccggcggc
     1621 ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc
     1681 ggactggtag ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt
     1741 gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt
     1801 gaagttcacc ttgatgccgt tcttctgctt gtcggccatg atatagacgt tgtggctgtt
     1861 gtagttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt
     1921 cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta
     1981 gttgccgtcg tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga
     2041 cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg gcgaagcact gcaggccgta
     2101 gccgaaggtg gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa
     2161 cttcagggtc agcttgccgt aggtggcatc gccctcgccc tcgccggaca cgctgaactt
     2221 gtggccgttt acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc
     2281 ctcgcccttg ctcaccatgg tggcggccgc tggcacggct ccggcctcgg cttcgtcttc
     2341 gacgagagtc tcgacctcga tctccgttcc gccgatgttc agtttggtgg ttttgcggat
     2401 atctccgtga atcaatatgt gctcgtggat caggacgcgc aggtagtgtc ccaacaaccc
     2461 ccaacaattt ttactcatca gatcaataat cgtgtgacct acggtggagc catagacgct
     2521 caggacgcca aaaccttcgg gccccaaaat gaagagaatt gggaacatac cccagctcac
     2581 gaaaaacagc catgccatgc cggtcacgac ctggcggcac cgaccctttg gcacagtatg
     2641 ataaccctcg atatatgctt tggcggcgtg aaaaaatgtg ttcgcgccat agcacaatcc
     2701 aagacaaaag aagatgactt taacatagcc ggttgccatg gcgctggtag ccccccacac
     2761 gatagtcccg atgtctgaga caaggagtcc catggttctc ctgctgtagt cgttgctcag
     2821 gccggtgagg ttgctcaggc ggataaggat gacaggacaa gtgagcagcc actctgcata
     2881 gcgcagccac tgcacccggt gtcctgtggc aaggtagagc atagagggat tcttaaactc
     2941 aaaaaagaac tcgagaatca ccttaaccat ttcaatggcg cacacataga tctcctccca
     3001 gccgcatgta gatttccagg tttggtaggc atagaacatc agcagcaaaa tgctgaatcc
     3061 tgctgcaagc cactgcagga catttgacgc ggtctgagcg ccgttcgtgc cgcgagattc
     3121 aatccatccg gcacagtaac attgatcctc agggaccagg acggacccgt tcaccaccac
     3181 aggattagta acgaacaaaa gttcgcgtcc gacggcagac aaagcgccgc catagtccat
     3241 ggtggctagc ataacttcgt ataaagtatc ctatacgaag ttatttgcct taacccagaa
     3301 attatcactg ttattcttta gaatggtgca aagaataact tcgtataatg tatgctatac
     3361 gaagttatga attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg
     3421 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     3481 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     3541 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     3601 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     3661 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     3721 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     3781 cggggaaatc atcgtccttt ccttggctgc tcgcctatgt tgccacctgg attctgcgcg
     3841 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     3901 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     3961 ccctttgggc cgcctccccg catcgatacc gagcgctgct cgagagatct acgggtggca
     4021 tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac
     4081 cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa
     4141 tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag
     4201 ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc
     4261 aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt tgttgggatt
     4321 ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac
     4381 catattggcc aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc
     4441 caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgattttgta
     4501 ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc
     4561 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     4621 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     4681 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     4741 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4801 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4861 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4921 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     4981 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     5041 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt
     5101 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     5161 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     5221 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     5281 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     5341 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     5401 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     5461 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5521 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5581 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5641 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5701 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5761 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5821 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5881 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5941 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6001 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6061 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6121 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6181 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6241 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6301 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6361 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6421 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6481 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6541 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6601 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6661 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6721 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     6781 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6841 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6901 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6961 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     7021 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7081 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7141 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7201 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7261 acatgt
//
