LOCUS       Exported                7315 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  High photocurrent, low-light sensitive channelrhodopsin (ChRger3) 
            for optogenetic activation with systemic delivery or for low-light 
            activation. Driven by the CAG promoter and double floxed..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-CAG-DIO(ChRger3-TS-YFP)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7315)
  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 7315)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7315
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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    1292..1325
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    1376..1409
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1421..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"
     protein_bind    complement(3300..3333)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    complement(3384..3417)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3442..4030
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3495..3515)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3913..3924)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4062..4538
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4261..4280)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4578..4718
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4578..4707
                     /label=AAV2 ITR
     rep_origin      4793..5248
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4880..4899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5090..5111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5265..5284)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5384..5406
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5444..5462)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5530..5634
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5635..6495
                     /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(5853..5872)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6666..7254
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7155..7174
                     /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 ggccgcgacc gcgccagcct
     2221 cggcctcgtc ctccaccagc gtctcgacct caatctcagt gccaccaatg ttcaatttgg
     2281 tggtcttgcg aatgtcgccg tggatgagga tatgctcgtg gattttgatg cgcaggaagt
     2341 gcccgagcag tccccaaata ttcttggaca gcaggtcggc gatggtgtgg ccaatagtgg
     2401 acccagccac gctcaggacg ccgaagccct cggggccgag gatgaacagg atggggaaca
     2461 taccccatga tacgaagaag agccaagcca tgccagtcac cacctggcga caccggccct
     2521 tcggcacggt gtggtacgcc tccacgtaga tgattcctgc gttgtagaat gtctgggtgc
     2581 cgtacaccag gccgatacag taaaacagcc acttcaccca cccagtggcc agagcggctg
     2641 tcacgcccat gcagatagtg cccaggtctg acaccagcag agccatggta cgcttattat
     2701 atgcctcact cagaccggtg atgttgctca gatggataag aatgacaggg catgtcagca
     2761 gccactcggc gtaacgaagc catggggtaa tgttgcctcc gtacaggaac agcatcgcag
     2821 gactgtcaaa ctcatggaaa tactcgatga tgaacttgat catctcgatc gtggccacgt
     2881 aaatctcctc ccagccgcaa gtagacttcc aggtctggta gccgtagaac atcaggcaga
     2941 gcgctgaaag cgcaaaagta atccactgca gaatgttggc agccaacttc tcggcatttg
     3001 ttccgttgga tttaagccaa gccaagcaga agcactggcc gttgttcggg atgcaaataa
     3061 cagaaccatt gttctcaaga gtgtatgagg tttggaagag catgcgctcg tgggcacggt
     3121 ggaaaacgta gtcggggcca tcctgagtcg cgaccggcac cgtcgcgtca ctgccagtcg
     3181 acttggtggg cttgtaggcg tccaccatca cgatgtgggc ggctcctgcg ctgccggccg
     3241 ccagcgccac tgccagcgct agggcaagaa gccatggcct ccgcgacatg gtggctagca
     3301 taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt
     3361 tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg aagttatgaa
     3421 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     3481 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3541 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     3601 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     3661 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     3721 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3781 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     3841 tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc
     3901 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     3961 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     4021 gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc
     4081 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc
     4141 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt
     4201 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
     4261 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct
     4321 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca
     4381 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca
     4441 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg
     4501 gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt
     4561 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4621 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4681 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     4741 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     4801 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4861 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4921 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4981 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     5041 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5101 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     5161 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5221 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     5281 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5341 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5401 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     5461 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5521 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5581 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5641 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5701 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5761 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5821 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5881 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5941 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6001 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6061 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6121 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6181 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6241 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6301 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6361 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6421 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6481 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6541 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6601 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6661 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6721 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6781 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6841 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6901 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6961 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7021 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7081 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7141 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7201 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7261 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
