LOCUS       Exported                7251 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV expression of Ef1a-driven, cre-dependent, hChR2 variant for 
            ultrafast optogenetic control..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a-DIO hChR2(E123T/T159C)-mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7251)
  AUTHORS   Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O'Shea DJ, Prakash R,
            Gunaydin LA, Hyun M, Fenno LE, Gradinaru V, Yizhar O, Deisseroth K
  TITLE     Principles for applying optogenetic tools derived from direct 
            comparative analysis of microbial opsins.
  JOURNAL   Nat Methods. 2011 Dec 18;9(2):159-72. doi: 10.1038/nmeth.1808.
  PUBMED    22179551
REFERENCE   2  (bases 1 to 7251)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7251
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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    1450..1483
                     /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    1534..1567
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1579..2286)
                     /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(1857..1876)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2126..2144
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2299..3225)
                     /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
                     PSMLYLATGHRVQWLRYATWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGCIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      3222..3231
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(3236..3269)
                     /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(3320..3353)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3378..3966
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3431..3451)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3849..3860)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3998..4474
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4197..4216)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4514..4654
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4514..4643
                     /label=AAV2 ITR
     rep_origin      4729..5184
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4816..4835)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5026..5047
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5201..5220)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5320..5342
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5380..5398)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5466..5570
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5571..6431
                     /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(5789..5808)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6602..7190
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7091..7110
                     /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 ccgccggtgg agtggcggcc
     1621 ctcggcgcgt tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg tgatgtccaa
     1681 cttgatgttg acgttgtagg cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt
     1741 cttgacctca gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct tgatctcgcc
     1801 cttcagggcg ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc ccatggtctt
     1861 cttctgcatt acggggccgt cggaggggaa gttggtgccg cgcagcttca ccttgtagat
     1921 gaactcgccg tcctgcaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa
     1981 gttcatcacg cgctcccact tgaagccctc ggggaaggac agcttcaagt agtcggggat
     2041 gtcggcgggg tgcttcacgt aggccttgga gccgtacatg aactgagggg acaggatgtc
     2101 ccaggcgaag ggcagggggc cacccttggt caccttcagc ttggcggtct gggtgccctc
     2161 gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc
     2221 catgtgcacc ttgaagcgca tgaactcctt gatgatggcc atgttatcct cctcgccctt
     2281 gctcacggcg gccgctggca cggctccggc ctcggcttcg tcttcgacga gagtctcgac
     2341 ctcgatctcc gttccgccga tgttcagttt ggtggttttg cggatatctc cgtgaatcaa
     2401 tatgtgctcg tggatcagga cgcgcaggta gtgtcccaac aacccccaac aatttttact
     2461 catcagatca ataatcgtgt gacctacggt ggagccatag acgctcagga cgccaaaacc
     2521 ttcgggcccc aaaatgaaga gaattgggaa cataccccag ctcacgaaaa acagccatgc
     2581 catgccggtc acgacctggc ggcaccgacc ctttggcaca gtatgataac cctcgatata
     2641 tgctttggcg gcgtgaaaaa atgtgttcgc gccatagcac aatccaagac aaaagaagat
     2701 gactttaaca tagccggttg ccatggcgct ggtagccccc cacacgatac acccgatgtc
     2761 tgagacaagg agtcccatgg ttctcctgct gtagtcgttg ctcaggccgg tgaggttgct
     2821 caggtggata aggatgacag gacaagtgag cagccaggtt gcatagcgca gccactgcac
     2881 ccggtgtcct gtggcaaggt agagcataga gggattctta aactcaaaaa agaactcgag
     2941 aatcacctta accatttcaa tggcgcacac atagatctcc tcccagccgc atgtagattt
     3001 ccaggtttgg taggcataga acatcagcag caaaatgctg aatcctgctg caagccactg
     3061 caggacattt gacgcggtct gagcgccgtt cgtgccgcga gattcaatcc atccggcaca
     3121 gtaacattga tcctcaggga ccaggacgga cccgttcacc accacaggat tagtaacgaa
     3181 caaaagttcg cgtccgacgg cagacaaagc gccgccatag tccatggtgg ctagcataac
     3241 ttcgtataaa gtatcctata cgaagttatt tgccttaacc cagaaattat cactgttatt
     3301 ctttagaatg gtgcaaagaa taacttcgta taatgtatgc tatacgaagt tatgaattcg
     3361 atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3421 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3481 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3541 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3601 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     3661 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3721 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     3781 cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct
     3841 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     3901 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     3961 ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc
     4021 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat
     4081 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag
     4141 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta
     4201 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg
     4261 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac
     4321 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct
     4381 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt
     4441 acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg
     4501 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4561 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4621 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4681 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4741 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4801 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4861 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4921 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4981 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     5041 aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc
     5101 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     5161 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     5221 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5281 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5341 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5401 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5461 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5521 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5581 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5641 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5701 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5761 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5821 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5881 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5941 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6001 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6061 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6121 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6181 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6241 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6301 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6361 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6421 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6481 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6541 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6601 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6661 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6721 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     6781 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6841 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6901 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6961 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7021 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7081 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7141 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7201 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
