LOCUS       sequence-247086-        7300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High photocurrent, low-light sensitive channelrhodopsin (ChRger2) 
            for optogenetic activation with systemic delivery or for low-light 
            activation. Driven by the CAG promoter and double floxed..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7300)
  AUTHORS   Bedbrook CN, Yang KK, Robinson JE, Mackey ED, Gradinaru V, Arnold FH
  TITLE     Machine learning-guided channelrhodopsin engineering enables 
            minimally invasive optogenetics.
  JOURNAL   Nat Methods. 2019 Oct 14. pii: 10.1038/s41592-019-0583-8. doi: 
            10.1038/s41592-019-0583-8.
  PUBMED    31611694
REFERENCE   2  (bases 1 to 7300)
  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: "Nat 
            Methods. 2019 Oct 14. pii: 10.1038/s41592-019-0583-8. doi: 
            10.1038/s41592-019-0583-8."
COMMENT     Sequence Label: pAAV-CAG-DIO(ChRger2-TS-YFP)
FEATURES             Location/Qualifiers
     source          1..7300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        210..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        515..792
                     /label=chicken beta-actin promoter
     primer_bind     1122..1144
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1206..1225
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(1292..1325)
                     /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(1376..1409)
                     /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(1421..2134,2135..2137))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1466..1487)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1815..1834
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2074..2095
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(3161..3199)
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     protein_bind    3285..3318
                     /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    3369..3402
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3427..4015
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3480..3500)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3898..3909)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4047..4523
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4246..4265)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4563..4703
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4563..4692
                     /label=AAV2 ITR
     rep_origin      4778..5233
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4865..4884)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5075..5096
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5250..5269)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5369..5391
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5429..5447)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5515..5619
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5620..5688,5689..6480)
                     /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(5838..5857)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6651..7239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7140..7159
                     /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 gcgtaacact agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
      541 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
      601 ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg
      661 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
      721 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
      781 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
      841 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
      901 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg
      961 tggctgcgtg aaagccttga ggggctccgg gagcgccggc aggaaggaaa tgggcgggga
     1021 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1081 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1141 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1201 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgcggcccaa
     1261 cggtaccgga tcctctagag tcgactccgg aataacttcg tataggatac tttatacgaa
     1321 gttatgcaga atggtagctg gattgtagct gctattagca atatgaaacc tcttaataac
     1381 ttcgtatagc atacattata cgaagttatg gcgcgcccta ttacttgtac agctcgtcca
     1441 tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct
     1501 cgttggggtc tttgctcagg gcggactggt agctcaggta gtggttgtcg ggcagcagca
     1561 cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt
     1621 cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca
     1681 tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt
     1741 cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact
     1801 tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga
     1861 cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc
     1921 gggcgaagca ctgcaggccg tagccgaagg tggtcacgag ggtgggccag ggcacgggca
     1981 gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc
     2041 cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg
     2101 gcaccacccc ggtgaacagc tcctcgccct tgctcaccac gttgatgtcg atctggtcca
     2161 gggggatgta ctcgccctcg ctggtgatcc tgctcttggc ggccgccact ttatcctcct
     2221 ctgtcacgaa ggccaggacg tccaccttgc ggcccagaaa ctgggagctc actggtctgc
     2281 gaatgtcgcc gtacataatg atatgctcgt ggattttgat gcgcaggaag tgcccgagca
     2341 gtccccaaat attcttggac agcaggtcgg cgatggtgtg gccaatagtg gacccagcca
     2401 cgctcaggac gccgaagccc tcggggccga ggatgaacag gatggggaac ataccccatg
     2461 atacgaagaa gagccaagcc atgccagtca ccacctggcg acaccggccc ttcggcacgg
     2521 tgtggtacgc ctccacgtag atgattcctg cgttgtagaa tgtctgggtg ccgtacacca
     2581 ggccgataca gtaaaacagc cacttcaccc acccagtggc cagagcggct gtcacgccca
     2641 tgcagatagt gcccaggtct gacaccagca gagccatggt acgcttatta tatgcctcac
     2701 tcagaccggt gatgttgctc agatggataa gaatgacagg gcatgtcagc agccactcgg
     2761 cgtaacgaag ccatggggta atgttgcctc cgtacaggaa cagcatcgca ggactggtaa
     2821 actcgaaata gatctccaga atcactttga tcagctccac ggagcacacg tacacctcct
     2881 cccagcccac gctggcctta taggcgtgcc agccgtagaa agccagaaac agggcagaca
     2941 ggaacacggc aaaccactgg aagcccctgg ctgtttcctg ctcccacagg gctcctctgc
     3001 tgtgcagcca agcggagcaa tagcacttgt cggctggtcc gcagacaaag tggtcttctg
     3061 gcaccacggc tcctttagcc agctcgtcga agccgttcat ggcgtagtga gacactgctg
     3121 cggcggctgt tccgtctgta gaagaagctg ctggggcgct cttggtgggc ttgtaggcgt
     3181 ccaccatcac gatgtgggca gaggctgttg ttgtgctggt aattccgcag aggaggagag
     3241 ccagcagcca agaagcggcg accagtctgc tcatggtggc tagcataact tcgtataaag
     3301 tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg
     3361 tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atgaattcga tatcaagctt
     3421 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     3481 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     3541 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     3601 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     3661 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     3721 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     3781 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     3841 ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     3901 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     3961 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     4021 taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
     4081 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
     4141 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
     4201 ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa
     4261 gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga
     4321 ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct
     4381 aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
     4441 cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa
     4501 ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc
     4561 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4621 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     4681 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     4741 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4801 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     4861 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4921 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4981 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5041 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5101 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     5161 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     5221 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5281 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5341 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5401 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5461 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5521 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5581 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5641 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5701 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5761 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5821 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5881 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5941 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6001 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6061 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6121 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6181 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6241 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6301 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6361 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6421 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6481 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6541 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6601 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6661 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6721 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6781 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6841 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6901 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6961 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7021 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7081 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7141 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7201 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7261 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
