LOCUS       sequence-354478-        7241 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-activated AAV expression of humanized ChR2 with H134R mutation 
            fused to mCherry for optogenetic activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7241)
  TITLE     Cre-activated AAV expression plasmids
REFERENCE   2  (bases 1 to 7241)
  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)-mCherry-W...
FEATURES             Location/Qualifiers
     source          1..7241
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      87..542
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(174..193)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     384..405
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(559..578)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     678..700
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(738..756)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        824..928
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(929..997,998..1789)
                     /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(1147..1166)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1960..2548
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2449..2468
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2610..2739
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2840..4018
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3071..4009
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3966..3986
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(4049..4082)
                     /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(4133..4166)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(4178..4885)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(4456..4475)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4725..4743
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(4898..5824)
                     /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      5821..5830
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    5835..5868
                     /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    5919..5952
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5977..6565
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6030..6050)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6448..6459)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6597..7073
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(6796..6815)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   join(7113..7241,1..12)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(7113..7241,1)
                     /label=AAV2 ITR
ORIGIN
        1 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
       61 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
      121 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
      181 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
      241 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
      301 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
      361 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
      421 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
      481 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
      541 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
      601 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
      661 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
      721 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
      781 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      841 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      901 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      961 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     1021 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1081 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1141 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1201 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1261 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1321 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1381 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1441 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1501 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1561 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1621 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1681 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1741 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1801 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1861 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1921 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1981 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2041 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2101 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2161 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2221 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2281 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2341 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2401 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2461 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2521 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2581 ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac
     2641 tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
     2701 gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcacg cgtaagcttt
     2761 gcaaagatgg ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt
     2821 cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
     2881 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
     2941 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     3001 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     3061 cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg
     3121 cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct
     3181 tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg
     3241 tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct
     3301 tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc
     3361 tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg
     3421 tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc
     3481 ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg
     3541 ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag
     3601 gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc
     3661 agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca
     3721 aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc
     3781 gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg
     3841 ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc
     3901 agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt
     3961 cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgagg
     4021 taccggatcc tctagagtcg actccggaat aacttcgtat aggatacttt atacgaagtt
     4081 atgcagaatg gtagctggat tgtagctgct attagcaata tgaaacctct taataacttc
     4141 gtatagcata cattatacga agttatggcg cgccctatta cttgtacagc tcgtccatgc
     4201 cgccggtgga gtggcggccc tcggcgcgtt cgtactgttc cacgatggtg tagtcctcgt
     4261 tgtgggaggt gatgtccaac ttgatgttga cgttgtaggc gccgggcagc tgcacgggct
     4321 tcttggcctt gtaggtggtc ttgacctcag cgtcgtagtg gccgccgtcc ttcagcttca
     4381 gcctctgctt gatctcgccc ttcagggcgc cgtcctcggg gtacatccgc tcggaggagg
     4441 cctcccagcc catggtcttc ttctgcatta cggggccgtc ggaggggaag ttggtgccgc
     4501 gcagcttcac cttgtagatg aactcgccgt cctgcaggga ggagtcctgg gtcacggtca
     4561 ccacgccgcc gtcctcgaag ttcatcacgc gctcccactt gaagccctcg gggaaggaca
     4621 gcttcaagta gtcggggatg tcggcggggt gcttcacgta ggccttggag ccgtacatga
     4681 actgagggga caggatgtcc caggcgaagg gcagggggcc acccttggtc accttcagct
     4741 tggcggtctg ggtgccctcg taggggcggc cctcgccctc gccctcgatc tcgaactcgt
     4801 ggccgttcac ggagccctcc atgtgcacct tgaagcgcat gaactccttg atgatggcca
     4861 tgttatcctc ctcgcccttg ctcacggcgg ccgctggcac ggctccggcc tcggcttcgt
     4921 cttcgacgag agtctcgacc tcgatctccg ttccgccgat gttcagtttg gtggttttgc
     4981 ggatatctcc gtgaatcaat atgtgctcgt ggatcaggac gcgcaggtag tgtcccaaca
     5041 acccccaaca atttttactc atcagatcaa taatcgtgtg acctacggtg gagccataga
     5101 cgctcaggac gccaaaacct tcgggcccca aaatgaagag aattgggaac ataccccagc
     5161 tcacgaaaaa cagccatgcc atgccggtca cgacctggcg gcaccgaccc tttggcacag
     5221 tatgataacc ctcgatatat gctttggcgg cgtgaaaaaa tgtgttcgcg ccatagcaca
     5281 atccaagaca aaagaagatg actttaacat agccggttgc catggcgctg gtagcccccc
     5341 acacgatagt cccgatgtct gagacaagga gtcccatggt tctcctgctg tagtcgttgc
     5401 tcaggccggt gaggttgctc aggcggataa ggatgacagg acaagtgagc agccactctg
     5461 catagcgcag ccactgcacc cggtgtcctg tggcaaggta gagcatagag ggattcttaa
     5521 actcaaaaaa gaactcgaga atcaccttaa ccatttcaat ggcgcacaca tagatctcct
     5581 cccagccgca tgtagatttc caggtttggt aggcatagaa catcagcagc aaaatgctga
     5641 atcctgctgc aagccactgc aggacatttg acgcggtctg agcgccgttc gtgccgcgag
     5701 attcaatcca tccggcacag taacattgat cctcagggac caggacggac ccgttcacca
     5761 ccacaggatt agtaacgaac aaaagttcgc gtccgacggc agacaaagcg ccgccatagt
     5821 ccatggtggc tagcataact tcgtataaag tatcctatac gaagttattt gccttaaccc
     5881 agaaattatc actgttattc tttagaatgg tgcaaagaat aacttcgtat aatgtatgct
     5941 atacgaagtt atgaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt
     6001 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     6061 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     6121 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     6181 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     6241 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     6301 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     6361 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg
     6421 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     6481 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     6541 atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt
     6601 ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc
     6661 ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct
     6721 ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct
     6781 gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca
     6841 ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg
     6901 gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt
     6961 tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc
     7021 ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt
     7081 tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac
     7141 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     7201 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc a
//
