LOCUS       sequence-191904-        7256 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 7256)
  TITLE     Cre-activated AAV expression plasmids
REFERENCE   2  (bases 1 to 7256)
  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..7256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..131
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        232..1410
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          463..1401
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1358..1378
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1441..1474)
                     /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(1525..1558)
                     /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(1570..2283,2284..2286,2287..2289))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1615..1636)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1964..1983
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2223..2244
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2286..2295
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2305..3231)
                     /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      3228..3237
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3242..3275
                     /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    3326..3359
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3384..3972
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3437..3457)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3855..3866)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4004..4480
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4203..4222)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4520..4660
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4520..4649
                     /label=AAV2 ITR
     rep_origin      4735..5190
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4822..4841)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5032..5053
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5207..5226)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5326..5348
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5386..5404)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5472..5576
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5577..5645,5646..6437)
                     /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(5795..5814)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6608..7196
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7097..7116
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt
       61 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      121 taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt ttaaacagag
      181 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
      241 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
      301 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
      361 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
      421 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
      481 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
      541 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
      601 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
      661 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
      721 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
      781 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
      841 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
      901 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
      961 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     1021 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     1081 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     1141 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     1201 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     1261 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     1321 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     1381 agtttttttc ttccatttca ggtgtcgtga ggtaccggat cctctagagt cgactccgga
     1441 ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg attgtagctg
     1501 ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac gaagttatgg
     1561 cgcgccctat tacttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa
     1621 ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg cggactggta
     1681 gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg
     1741 gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct tgaagttcac
     1801 cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt tgtagttgta
     1861 ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat
     1921 gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc
     1981 gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa
     2041 gtcgtgctgc ttcatgtggt cggggtagcg ggcgaagcac tgcaggccgt agccgaaggt
     2101 ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt
     2161 cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt
     2221 tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt
     2281 gctcaccatg gtggcggccg ctggcacggc tccggcctcg gcttcgtctt cgacgagagt
     2341 ctcgacctcg atctccgttc cgccgatgtt cagtttggtg gttttgcgga tatctccgtg
     2401 aatcaatatg tgctcgtgga tcaggacgcg caggtagtgt cccaacaacc cccaacaatt
     2461 tttactcatc agatcaataa tcgtgtgacc tacggtggag ccatagacgc tcaggacgcc
     2521 aaaaccttcg ggccccaaaa tgaagagaat tgggaacata ccccagctca cgaaaaacag
     2581 ccatgccatg ccggtcacga cctggcggca ccgacccttt ggcacagtat gataaccctc
     2641 gatatatgct ttggcggcgt gaaaaaatgt gttcgcgcca tagcacaatc caagacaaaa
     2701 gaagatgact ttaacatagc cggttgccat ggcgctggta gccccccaca cgatagtccc
     2761 gatgtctgag acaaggagtc ccatggttct cctgctgtag tcgttgctca ggccggtgag
     2821 gttgctcagg cggataagga tgacaggaca agtgagcagc cactctgcat agcgcagcca
     2881 ctgcacccgg tgtcctgtgg caaggtagag catagaggga ttcttaaact caaaaaagaa
     2941 ctcgagaatc accttaacca tttcaatggc gcacacatag atctcctccc agccgcatgt
     3001 agatttccag gtttggtagg catagaacat cagcagcaaa atgctgaatc ctgctgcaag
     3061 ccactgcagg acatttgacg cggtctgagc gccgttcgtg ccgcgagatt caatccatcc
     3121 ggcacagtaa cattgatcct cagggaccag gacggacccg ttcaccacca caggattagt
     3181 aacgaacaaa agttcgcgtc cgacggcaga caaagcgccg ccatagtcca tggtggctag
     3241 cataacttcg tataaagtat cctatacgaa gttatttgcc ttaacccaga aattatcact
     3301 gttattcttt agaatggtgc aaagaataac ttcgtataat gtatgctata cgaagttatg
     3361 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     3421 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     3481 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     3541 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     3601 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     3661 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     3721 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     3781 catcgtcctt tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct
     3841 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     3901 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     3961 ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc tacgggtggc atccctgtga
     4021 cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt
     4081 cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg
     4141 gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg
     4201 ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc
     4261 ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat tccaggcatg
     4321 catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc
     4381 caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct
     4441 gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt aggtaaccac
     4501 gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     4561 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     4621 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     4681 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     4741 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4801 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4861 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4921 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     4981 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5041 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     5101 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5161 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     5221 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5281 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5341 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     5401 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     5461 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5521 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5581 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5641 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5701 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5761 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5821 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5881 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5941 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6001 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6061 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6121 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6181 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6241 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6301 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6361 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6421 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6481 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6541 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6601 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6661 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6721 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6781 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6841 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6901 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6961 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     7021 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7081 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     7141 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7201 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatg
//
